L1000 Standard Operating Procedure
L1000 Standard Operating Procedure May 2014 Connectivity Map Platform The Broad Institute
Overview
Protocol: Transcript Capture and Ligation Mediated Amplification In reverse transcription, mRNA transcripts from whole cell lysate or purified RNA are isolated by capturing the polyA tail of the transcript on the surface of a polyT coated turbocapture plate. Then the reverse transcriptase enzyme is used to build a cDNA complement to this polyT tail. Upstream and downstream probes containing gene specific, LUA “barcode” tags, and universal primer sequences are then added to the mix. The probes anneal and ligate to the gene specific region of the transcript. Finally, universal biotinylated primers are added to the mix and PCR amplified. The final product is biotinylated, barcoded PCR amplicon of 104-nt length.
Transcript Capture (CAP) Purpose
Immobilize mRNA from lysed cells onto the walls of the Turbocapture plate, by providing a dT coated surface which binds the poly-A tail of each mRNA transcript. RELATED PROTOCOL This step requires materials produced via the related protocol Cell Based Assay. Alternately, mRNA acquired by some other method can be used.
IMPORTANT Prepare materials and assets in pre-PCR and RNAse-free environment.
Preparation 1. 2. 3. 4.
Thaw LYSIS plates at 4°C Thaw Reference Total RNA M07 on ice Prepare CyBio tips according to plate layout for lysis transfer Label Turbocapture plates
Materials TCL buffer M01 MicroAmp seals M02 CyBio 384-well tips M03 Turbocapture plates M04 1.5ml eppendorf tubes M05
Assets Vortex A12 Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13
Reagent Mix ID
MIX01
Name MCF7 Total RNA master mix
Step
Composition
CAP
19.7μl TCL Lysis Buffer M01 0.3μl MCF7 Total RNA M07
Volume/Well
20μl
Use Keep on ice, incubate at room temp for 5m before using
Procedure 1. Label each new Turbocapture plate with information listed on the corresponding cell lysate source plate (compound set, cell line, date, plate #). Include also current date and initials. 2. Document names of plates being taken through RT on tracking sheet. 3. Centrifuge thawed cell lysate (LYSIS) plates for 1min @1000RPM. Keep plates on ice until ready to transfer. 4. Place LYSIS plate at Position 1 of CyBio and its corresponding labeled Turbocapture plate (CAP) at Position 2.
TIP: Check orientation of CAP and LYSIS plates.
5. Load CyBio 384 tip cartridge. TIP: Ensure CyBio tips have been arranged in rack to transfer only relevant lysate from LYSIS plate as specified by the ‘Plate Format’. Take care not to let any lysate get into control wells.
6. Transfer 20 μl lysate from LYSIS to CAP plate using CyBio program: 384 Lysate Capture . 7. Repeat steps 1-5 until all LYSIS plates have been transferred. 8. Prepare Reference Total RNA controls. 1. Mix Total RNA master mix MIX01 for each cell line included in ‘Plate Format’, using 1.5 ml Eppendorf tubes. Flick Eppendorf tubes to mix and incubate at room temperature for 5 min. IMPORTANT: Keep total RNA on ice, limit freeze/thaw cycles to 4, and return to -80C storage as soon as possible. Do not try to mix directly in wells.
2. Document REF RNAs prepared and their F/T# on tracking sheet. 3. Pipette 20μl of Total RNA master mix to corresponding control wells of all CAP plates according to ‘Plate Format’. 9. Seal plate with MicroAmp seal. 10. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for empty or low volume wells and mark on tracking sheet.
11. Incubate at Room Temperature for 1 hour. 12. Proceed directly to preparation for Reverse Transcription (FIRST).
Reverse Transcription (FIRST) Purpose Synthesize an immobolized cDNA that is complementary to the mRNA by using the immobilized dT as the primer and the captured mRNA as the template. IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 5x M-MLV RT buffer M18 2. Thaw 25mM dNTPs M19 3. Leave M-MLV RT Enzyme M20 at -20°C
Materials Plates from CAP step DEPC treated water M17 kept at -20° CyBio 384-well tips M03 Reagent reservoir M16
Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13 PrePCR thermocyclers A02 20° freezer, 4th floor A04
Reagent Mix ID
MIX02
Name
RT master mix
Step
RT
Composition 3.7μl DEPC treated water M17 1μl 5X M-MLV RT Buffer M18 0.1μl 25mM dNTPs M19 0.2μl M-MLV RT enzyme M20
Volume/Well
5μl
Use
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare FIRST master mix without enzyme MIX02 on ice IMPORTANT: Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out lysate from CAP plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of CAP plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. 3. Transfer 5μl of master mix from the reservoir to destination CAP plate using CyBio program: 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load CAP plate in Position 1 and place RT master mix source plate in Position 2. 3. Run CyBio program. 4. Repeat until all CAP plates have been processed. TIP: Do not let the CAP CAP plates sit dry for too long. Do not use plate stacker. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. 5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate in pre-PCR thermocycler using program: 37for90. STOP POINT: If procedure will be continued at a later time, store CAP CAP plates at -20°C, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Reverse Transcription was completed.
7. Proceed directly to preparation for Probe Anneal (ANNL).
Probe Anneal (ANNL) Purpose Bind probe pairs targeting specific sequences for genes of interest to target cDNA which corresponds to them. These sequences are located adjacent to each other on the cDNA template and contain LUA tags and universal primer sites to be used in later steps. IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 10X DNA ligase buffer M23 2. Thaw probe pool M22 at room temperature 3. Check FIRST plates for evaporated wells
Materials Plates from FIRST step CyBio 384-well tips M03 Nuclease free water M21 Reagent reservoir M16 Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Vortex A12 Plate heater A30 Benchtop centrifuge A09 CyBio Variable 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
MIX03
Name
Probe anneal master mix
Step
ANNL
Composition 4.22μl Nuclease free water M21 0.28μl "1000" probe pair mix M22 0.5μl 10X Taq ligase buffer M23
Volume/Well
5μl
Use
Keep at RT, warm probes before adding
Procedure 1. Prepare ANNL master mix without probes MIX03 at room temperature. IMPORTANT: Add probes just before beginning procedure.
TIP:Warm probes to 50 C for 10 to 20 seconds before adding.
1. Vortex to mix and pour into reagent reservoir at room temperature. 2. Spin out master mix from FIRST plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of FIRST plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 5μl of master mix from reservoir to destination FIRST plate using CyBio program 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load FIRST plate in Position 1 and load Probe Anneal master mix in Position 2. 3. Run CyBio program 4. Repeat until all FIRST plates have been processed. TIP:Do not let the FIRST plates sit dry for too long. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate overnight in Pre PCR thermocycler using program probebind6hramp.
STOP POINT: If procedure will be continued at a later time, store ANNL ANNL plates at -20°C in the 4th floor freezer A04, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Anneal was completed.
7. Proceed directly to preparation for Probe Ligation (LIG).
Probe Ligation (LIG) Purpose Ligate together the two probes of each probe pair to form a single strand comprising the entire 40 nucleotide gene specific sequence flanked by a LUA tag and T3 & T7 primer site. IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 10X Taq DNA ligase buffer M23 2. Check ANNL plates for evaporated wells 3. Note hold duration of ANNL plates on tracking sheet
Materials Plates from ANNL step CyBio 384-well tips M03 Nuclease free water M21 Taq DNA ligase enzyme M20 kept at -20°C Reagent reservoir M16 Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Vortex A12 Plate heater A30 Benchtop centrifuge A09 CyBio Variable 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
MIX04
Name
Ligation master mix
Step
Composition
LIG
4.4375μl Nuclease free water M21 0.5μl 10X Taq ligase buffer M23 0.0625μl Taq DNA ligase M24
Volume/Well
5μl
Use
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare LIG master mix without enzyme MIX04 on ice. IMPORTANT:Keep enzyme at -20 C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out master mix from ANNL plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of ANNL plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 5μl of master mix from reservoir to destination ANNL plate using CyBio program 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load ANNL plate in Position 1 and load Probe Anneal master mix in Position 2. 3. Run CyBio program 4. Repeat until all ANNL plates have been processed. TIP:Do not let the ANNL plates sit dry for too long. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate overnight in Pre PCR thermocycler using program: probelig384. TIP:The program holds at 45°C for one hour, 65°C for 10 minutes, then holds overnight at 4°C.
STOP POINT: If procedure will be continued at a later time, store LIG plates at -20°C in the 4th floor freezer A04 , sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Ligation was completed.
7. Proceed directly to preparation for PCR Amplification step (PCR).
PCR Amplification (PCR) Purpose Denature ligated probe pairs from their cDNA templates, and amplify gene transcripts of interest exponentially from their cT3 and cT7 primer sites. The final products are same length, biotinylated, barcoded PCR amplicons. IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 25 mM dNTPs M19 with T3 M27 & T7 M38 primers 2. Thaw 25mM MgCl2 M26 3. Thaw 10X PCR buffer M25 4. Check LIG plates for evaporated wells
Materials Plates from LIG step Nuclease free water M21 HotStarTaq M29 kept at -20°C CyBio 384 well tips M03 Reagent reservoir M16 Super rag M12 Aluminum foil M13 MicroAmp seal M02
Assets Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
Name
Step
Composition
Volume/Well
Use
12.768μl Nuclease free water M21 1.5μl 10X PCR buffer M25 0.51μl MgCl2 M24 MIX05
PCR master mix
PCR
0.096μl 25mM dNTPs M19 0.015μl T3 primer 100μM M27 0.015μl T7 primer 100μM M28 0.096μl HotStarTaq M29
15μl
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare PCR master mix without enzyme MIX05 on ice. IMPORTANT:Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out LIG plates. 1. Remove centrifuge plate holders from rotor. 2. Remove seal of LIG plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Place wrapped plate inverted into plate holder. TIP:See Figure 5 for configuration of plate ready to spin out.
6. Spin for 1min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 15 μl of master mix from source plate to destination PCR plate using CyBio program 15 μl PCR addition. 1. Load new CyBio 384 tip cartridge TIP: Use the same tips for all transfers in this step.
2. Load LIG plate in position 1 and place PCR master mix reagent reservoir in position 2. 3. Run CyBio program 4. Repeat until all LIG plates have been processed 4. Seal the plates with MicroAmp seal IMPORTANT: Must seal plates very tightly before cycling. Press down firmly and edge wells tightly with a pen.
5. Centrifuge plates for 15 seconds at 1000RPM to settle liquid in wells
TIP:Check for debris in wells and mark on tracking sheet.
6. Incubate in 7th floor Post PCR Thermocycler using program PCR29 ALT 1M CYC. TIP: The program brings the material to 92°C, then does 29 cycles as follows: 92°C for 1 minute, 60°C for one minute, 72°C for one minute. After cycling, it holds for 5 minutes at 72°C, ad then holds at 10°C.
STOP POINT: If procedure will be continued at a later time, store PCR PCR plates at -20°C in the 7th floor freezer A05, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date PCR Amplification was completed.
7. Proceed directly to preparation for Hybridization (HYB)
Hybridization (HYB) Purpose Bind each amplified gene transcript to its corresponding Flexmap tag encoded bead in a detection plate. The tag-duo version of this protocol combines the 2 sets of 500 fluorescent beads at different proportions to create a single 1000-plex detection set instead of maintaining two separate subsets. RELATED PROTOCOL: This step requires materials produced via the related protocol Bead Preparation.
IMPORTANT: Prepare materials and assets in a Post-PCR environment.
Preparation 1. Thaw positive amplicon M74 2. Check PCR plates for evaporated wells
Materials Plates from PCR step Coupled magnetic beads set A M32A Coupled magnetic beads set B M32B 1.5X TMAC MIX09 Reagent reservoir M16 Nanoscreen tips M30 Pink microseal M34 TE buffer M63
Assets 7th floor benchtop centrifuge A10 7th floor sonicator A17 7th floor vortex A16 Biomek A27
Matrix electronic pipette A14 Post PCR thermocycler A03
Reagent Mixes ID
Name
Coupled bead mix
MIX07
MIX08
1X TMAC
MIX09
1.5X TMAC
Step
Composition
Volume/Well
Use
HYB
M32A Bead set A (includes controls at 0.5X) M32B Bead set B (includes controls at 0.5X) MIX09 1.5X TMAC
21μl
Store at 4°C
HYB
150ml TMAC M31 1.25ml 20% Sarkosyl M33 12.5ml Tris-HCl M35 2.0ml 0.5M EDTA M36 84.25ml DDW M40
250ml
Store at room temperature
HYB
225ml TMAC M31 1.88ml 20% Sarkosyl M33 18.75ml Tris-HCl M35 3ml 0.5M EDTA M36 1.37ml DDW M40
250ml
Store at room temperature
Procedure 1. Label each new HYB plate with information listed on the corresponding PCR source plate. Include also current date and initials. 2. Document names of plates being taken through hybridization on tracking sheet. 3. Prepare Coupled Bead Mix MIX07. TIP:Sonicate and vortex beads to ensure uniform dispersal.
Add 21μl bead mix into plates using Biomek program cmap prep/ bead mix addition. Reconfigure Biomek deck for PCR amplicon transfer. Run program. Add positive amplicon control 1. Dispense 5μl of positive amplicon into well B01 8. Seal plates tightly with Microseal A. 9. Incubate overnight at 45°C in 7th floor Post PCR Thermocycler using program hybridize. 4. 5. 6. 7.
TIP: This program includes a 95C denaturing step before the 45 C hold.
10. Proceed directly to preparation for SAPE Stain (SAPE).
Sape Stain (SAPE) Purpose Add a fluorescent label to each amplified gene bound to a given bead. This will allow the Luminex Flexmap3D system to quantitatively measure the amount of amplicon for each gene. IMPORTANT:Prepare materials and assets in a Post-PCR environment.
Preparation 1. Check HYB plates for evaporated wells 2. Ensure Biomek waste is empty and DDW source is full
Materials Plates from HYB step DDW M40 70% EtOH M41 1X TMAC MIX08 SAPE M37 Wash buffer A MIX12 Wash buffer B MIX13 384 well plates M38 Nanoscreen tip box cover M30
Assets 7th floor benchtop centrifuge A10 Nanoscreen A18 Biomek A27 Vortex A12 Matrix electronic pipette A14 45°C incubator A11 384 well magnets A29
Reagent Mix ID MIX06
Name SAPE master mix
Step
Composition
SAPE
9.7μl 1X TMAC MIX08 0.3μl SAPE M37
Volume/Well 10μl
Use Keep at room temperature and avoid exposure to light
Procedure TIP: Begin FlexMap 3D scanner warmup for detection step before beginning SAPE stain in order to save time.
1. Spin down HYB plates at 3000 rpm for 3 minutes. TIP: Check that beads have pelleted in the bottom of plate wells.
2. Load Biomek program file cmap cleanup that corresponds to the correct number of plates. 3. Place reagents and plates on deck as follows:
4. Run Biomek program TIP: The program will run the buffer A and B washes, and then pause for the SAPE addition.
5. Prepare SAPE master mix MIX06 6. The program will pause. Replace buffer B with reservoir of SAPE master mix and click OK to continue. TIP: Program will add 10µl SAPE mix and then pause.
7. Remove plates from Biomek deck. 8. Seal HYB plates with MicroSeal A. 9. Vortex HYB plates to mix beads and SAPE IMPORTANT: Ensure that bead pellets are fully dispersed.
10. 11. 12. 13. 14.
Incubate HYB plates in 45°C incubator for 10 minutes. Spin down plates at 3000RPM for 1 minute Remove seals and place HYB plates back on appropriate deck locations. Select OK to continue running program Seal plates with MicroSeal foil.
STOP POINT: If procedure will be continued at a later time, store SAPE SAPE plates for up to one week at 4°C in the 7th floor refrigerator A25, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date SAPE Stain was completed.
15. Proceed directly to preparation for Detection (DET) step.
Detection (DET) Purpose Read each detection plate with the Luminex FlexMap 3D System, which will determine the identity of each bead based on the internal dye ratio, and the amount of amplicon bound to it by the fluorescence of the SAPE. IMPORTANT: Prepare materials and assets in a post PCR environment.
Materials Plates from SAPE step
Assets FlexMap 3D scanners A07
Procedure 1. Prepare FlexMap 3D scanners 1. Turn on Flexmap 3D Scanners and Computers. 2. Login to Windows 3. Login to XPonent software 4. Eject tray, and fill position RA1 with DDW, RA2 with 70% EtOH 5. Run maintenance program Daily Instrument Startup TIP: Scanner will take 30 minutes to warm up.
2. 3. 4. 5. 6. 7. 8.
Open Batch tab. Choose New Batch from Existing Protocol. Select protocol CMAP FINAL. Eject plate tray. Load SAPE plate with correct orientation Run batch. When complete, run maintenance program Daily Instrument Shutdown.
Probe Pair Preparation Probe Collapse Purpose Collapse probes from 100uM stocks into a single tube.
Preparation
1. Thaw 100μM probe stocks M54 2. Label probe aliquot tubes
Materials Cryovials M67 Reagent reservoir M16
Assets Water bath A26 Vortex A12 20°C freezer, 4th floor A04
Procedure 1. 2. 3. 4. 5.
Label probe aliquot tubes with probe pool version, date collapsed, and initials Vortex probe plates. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. Uncap probe plates. Aspirate 2μl from each probe well using a multichannel pipette, and dispense into a reagent reservoir. TIP: Change tips each time you aspirate probes to avoid contamination.
6. 7. 8. 9. 10.
Transfer probe collapse set into a Falcon tube. Vortex collapsed probe set. Heat probes at 50C in a water bath within a beaker of distilled water for 30 seconds. Aliquot probe mix in 500μl volumes into Cryovials. Store probe aliquots in -20°C freezer.
Bead Preparation Bead Barcode Coupling (BBC) Purpose Bind the probe (and therefore target gene) specific LUA tag to each individual bead. To complete a full compiled detection, three detection plates are each coupled to different LUA tags. Calibration genes appear across all three detection plates.
Preparation 1. Prepare MES buffer MIX14 2. Thaw LUAs M56
Materials 50 ml Falcon tube M66 EDC M58 DDW M40 MES M62 Luminex magnetic microspheres M72 Collection microtubes M54
96-well deep well plate M60 Reservoir plates M61 CyBio 96 well tips M57 Reagent reservoir M16 Microseal foil M39 96 well magnets A28
Assets CyBio 96 A21 Combi A31 Sonicator A15 Vortex A12 Centrifuge A09
Reagent Mix ID
MIX14
Name
MES Coupling Buffer
Step
Composition
BBC
4.88g MES M62 5 drops 5M NaOH M71 249.95ml DDW M40
Volume/Well
250ml
Use
Bring pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C
Procedure IMPORTANT: IMPORTANT: Complete Complete this this process process as as quickly quickly as as possibly possibly to to minimize minimize bead bead exposure exposure to to light. light.
1. Aliquot 500 ml of Luminex beads in ascending order into racks of 96 deep well plates. TIP: Sonicate and vortex Luminex beads thoroughly in their stock bottles before aliquoting. Label plates with correct bead numbers.
2. Centrifuge plates at 3000 rpm for 1 minute. 3. Remove buffer from bead plates using CyBio program Remove 500 from 2 DW. 1. Place 96 well magnets on Position 1 and Position 3 of the CyBio. 2. Place 96 deep well reservoir in Position 2 to collect waste. 3. Place a bead coupling plate on each magnet and let sit one minute. TIP:The beads will pellet tightly inside the magnetic ring.
4. Load 96 well CyBio tip cartridge corresponding to the bead set. 5. Run CyBio program 4. Add MES using Combi program 65 ul, 96 DW (44 mm) Full Plate. 1. Bring bead plate to Combi. 2. Load large cassette into Combi. 3. Place Combi intake into MES buffer and prime 4. Run program 5. Add LUA tags 1. Thaw LUA plates 2. Vortex and spin down LUA plates in centrifuge (1000 rpm for 1 min).
3. Aspirate 2.5 μl of LUA from source plate. 4. Dispense 2.5 μl of LUA into its designated well in the bead plate, referring to the LUA-Bead layout map. TIP:Confirm that LUA is added to each well.
5. Repeat for all LUA-bead pairs 6. Add EDC using Combi program 65 ul, 96 DW (44 mm), Full Plate. 1. Bring all bead plates to Combi. 2. Load Small Combi cassette. 3. Mix 50 ml batch of 10 mg/mL EDC in double distilled water (DDW) and pour into reagent reservoir. 4. Run program for each plate IMPORTANT: Complete this process in 5 minutes if possible
5. 6. 7. 8.
Cover with MicroSeal foil Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. Incubate in dark at room temperature for 30 minutes. Repeat EDC addition and incubation for a second time with a new batch of EDC solution. Do not remove EDC from first addition. 7. Proceed directly to Bead Coupling Wash.
Bead Coupling Wash and Bead Collapse Purpose Remove all unbound LUA tags from bead mix to prevent non-specific binding of LUA tags to beads, then collapse beads into sets.
Preparation 1. Prepare Bead Wash Buffer I MIX10 2. Prepare Bead Wash Buffer II MIX11
Materials DDW M21 Tween-20 M64 SDS M65 TE buffer M63 96-well deep well plate M60 CyBio 96 well tips M57 Microseal foil M39 Reservoir plates M61 96 well bead magnet A28
Assets Vortex A12 CyBio 96 A21
Reagent Mixes
ID
Name
MIX10
Bead Wash Buffer I
MIX11
Bead Wash Buffer II
Step
Composition
Volume/Well
Use
BBC
50μl Tween-20 M64 249.95ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
BBC
2.5ml SDS 10% M65 247.5ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
Procedure 1. Wash beads with Bead Wash Buffer I using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash Buffer I and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 2. Wash beads with Bead Wash Buffer II using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash Buffer II and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 9. Vortex plate gently to disperse beads 3. Wash beads with TE Buffer using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash TE and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 9. Vortex plate gently to disperse beads 4. Collapse bead set into a single tube using CyBio. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Aspirate each well of the bead plate, and collapse into a 50 ml Falcon tube. 6. Dispense 50 ul of TE into the first column of the aspirated bead plate, then mix and transfer to the next column. TIP:Take a second pass through the bead plate to recover as many beads as possible.
7. Add TE with recovered beads to Falcon tube.
Assets ID
Name
Model/Vendor
Description
Unit/Location
A01
4th floor liquid handler
CyBio Vario
PERT, prePCR liquid handling, master mix additions
CyBio 384, 4th floor
A02
PrePCR thermocycler
Thermo MBS Satellite 384
FIRST - LOG, prePCR
4th floor
A03
PostPCR thermocycler
Themro MBS Satellite 384
PCR - HYB, postPCR
7th floor
A04
Freezer, -20°C
Sanyo HF-5015
FIRST-LIG, Store LMA reagents
CMAP -20°C pre, 4th floor
A05
Freezer −20°C
Sanyo MDF-U537
HYB, Store amplified PCR product
CMAP -20 post, room 7177
A06
Freezer −80°C
Sanyo MDF-U75VC
LYSIS, Store cell lysate pre-LMA
CMAP -80°C room 4051
A07
FlexMap 3D
Luminex
DET, Detect LMA product
n=9, Room 7177
A08
6 nL slot pin tool
V&P Scientific
PERT, Add small molecule
Pin tool, 4th floor
A09
Benchtop centrifuge
Eppendorf 5810
FIRST – LIG, Spin down/out mixes
prePCR, 4th floor
A10
Benchtop centrifuge
Eppendorf 5810
HYB – WASH, Spin down/out mixes
postPCR, room 7177
A11
Incubator 45°C
VWR Model 1535
SAPE, Incubate during SAPE stain
CMAP incubator, room 7177
A12
Vortex
VWR G560
CAP – ANNL, mix liquids
CMAP vortex, 4th floor
A13
prePCR multichannel
Thermo Matrix 16 Channel
CAP - PCR, add mixes to plates
CMAP prePCR matrix, 4th floor
A14
postPCR multichannel
Thermo Matrix 16 Channel
HYB - WASH, add mixes to plates
CMAP postPCR matrix, room 7177
A15
4th floor sonicator
VWR Ultrasonic, 2L
BBC, disperse settled bead
CMAP sonicator, 4th foor
A16
Vortex
VWR G560
HYB - SAPE, mix liquids
CMAP 7th floor vortex, room 7177
A17
7th floor sonicator
VWR Ultrasonic, 2L
HYB, disperse settled bead
CMAP sonicator, room 7177
A18
Liquid handler
Nanoscreen
HYB, liquid transfer postPCR
Nanoscreen 5th floor, Multimek 5th floor
A19
37°C Incubator
Thermo Scientific 3110
PLATE, cell culture incubator
CMAP TC incubator, 4th floor
A20
Plate rocker
Barnstead 4631
LYSIS, plate rocker
CMAP plate rocker, 4th floor
A21
CyBio 96
CyBio96
BBC, 96 well liquid transfer
CyBio96, 4th floor
A22
Cell counter
Beckman Coulter ViCell XR
PLATE, cell viability counter
ViCell, 4th floor TC room
A23
TC centrifuge
Beckman Coulter Allegra X-15R
PLATE, tissue culture centrifuge
TC centrifuge, 4th floor TC room
A24
TC multichannel
Ranin 1200ul pipette
PLATE, cell plating multichannel pipette
Ranin pipette, 4th floor TC room
A25
4°C fridge
SciCool SCGP210W1AREF
HYB, storage of post-hyb plates
4°C fridge, room 7177
Fisher Scientific
A26
Water bath
Isotemp 215
PROBE, prove denaturing
Water bath, 4th floor
A27
7th floor liquid handler
Biomek NX
SAPE - WASH, liquid transfer and wash postPCR
Biomek, room 7177
A28
96 well magnets
Agencourt
BBC, hold down magnetic bead
AGN#00219 | 4th floor
A29
384-well magnets
Dexter Magnetic
WASH, hold down magnetic bead
2501007 | Room 7177
A30
Plate Warmer
A31
Combi
A32
Janus liquid handler
A33
BL2+ centrifuge
A34
Electronic pipette
A35
Aspirating pipette
A36
BL2+ cell culture incubator
Reagents from external vendors ID
Name
Vendor
Catalog #
Process
M01
TCL Lysis Buffer
Invitrogen
15596-018
CAP, LYSIS
M02
MicroAmp Seal
Applied Biosystems
4306311
CAP - PCR
M03
CyBio 384 tips
CyBio
OL 3800-25-513-N
CAP - PCR
M04
384 well Turbocapture plate
Qiagen
72271
CAP - PCR
M05
1.5ml microtubes
ISC Bioexpress
C-3217-1
CAP
M06
HeLa S3 Total RNA
Ambion
Am7852
CAP
M07
MCF7 Total RNA
Ambion
AM7846
CAP
M08
HL60 Total RNA
Ambion
AM7836
CAP
M09
K562 Total RNA
Ambion
AM7832
CAP
M10
PC3 Total RNA
Ambion
AM7834
CAP
M11
MDA-MB-453 Total RNA
Ambion
AM7876
CAP
M12
Super Rag
Mercantile Development
93101
FIRST – LIG
M13
Aluminum Foil
Goldman Paper
1217
FIRST – LIG
M14
384-well plate
Eppendorf
951020737
FIRST – HYB
M15
15 ml Falcon tubes
BD Biosciences
352096
FIRST-STAIN
M16
Reagent Reservoir
Corning
4870
M17
DEPC treated water
Ambion
AM9915
FIRST
M18
5x M-MLV RT buffer
Promega
M2505
FIRST
M19
25 mM dNTPs
Invitrogen
10297018
FIRST, PCR
M20
M-MLV RT enzyme
Promega
M2505
FIRST
M21
Nuclease free water
Qiagen
XXXXX
ANNL - PCR
FIRST - STAIN
M22
Probe Dilution Plates
In-house
Custom
ANNL
M23
10x Taq ligase buffer
NEB
M0208L
ANNL, LIG
M24
Taq DNA ligase
NEB
M0208S
LIG
M25
10x PCR buffer
Qiagen
203443
PCR
M26
Magnesium Chloride
Qiagen
203443
PCR
M27
T3 primer
IDT
-
PCR
M28
T7 primer
IDT
-
PCR
M29
HotStarTaq
Qiagen
203443
PCR
M30
Nanoscreen tips 30 ul
Nanoscreen
30ulnc
HYB – WASH
M31
TMAC
Sigma
T3411-1L
HYB, WASH
M32A
Coupled Beads
In-House
Custom
HYB
M32B
Coupled Beads
In-House
Custom
HYB
M33
20% Sarkosyl
Sigma
L-9150
HYB
M34
MicroSeal
BioRad
MSA5001
HYB
M35
1M Tris-HCl, pH 8.0
Sigma
T-3038
HYB
M36
0.5M EDTA, pH 8.0
Gibco
15575-038
HYB
M37
SAPE
Invitrogen
S866
SAPE
M38
384-well plate
Costar
3456
SAPE
M39
Microseal F Foil
BioRad
MSF1001
DET
M40
Distilled Water
In House
-
DET
M41
70% Ethanol
In House
-
DET
M42
384-well cell culture plate
Corning
3570
PLATE
M43
PBS
MediaTech
21-040-CV
PLATE
M44
Trypsin EDTA
Invitrogen
25200-114
PLATE
M45
Vi-Cell Sample Vials
Beckman Coulter
383721
PLATE
M46
10cm cell culture petri dish
BD Falcon
353003
PLATE
M47
RPMI 1640 Media
Fisher Scientific
MT-10-040-CM
PLATE
M48
DMEM Media
Invitrogen
11995073
PLATE
M49
DMSO
Sigma
D2650-100ML
PERT
M50
Methanol
Fisher
A412-4
PERT
M51
1000x CAS compound plate
ChemBio
Custom
PERT
M52
Deep well 384-well plate
Costar
3965
LYSIS
M53
Luminex Carboxylated Microspheres
Luminex
-
BBC
M54
Collection Microtubes
Qiagen
19560
BBC
M55
100 µM stock probe plates
IDT
Custom
BBC
M56
LUA 96-well stock plates
IDT
Custom
BBC
M57
CyBio 96 well tips
CyBio
OL 3800-25-559-N
BBC
M58
EDC
Pierce Biosciences
22980
BBC
M59
96-well plate
Axygen
321-21-051
BBC
M60
96-well deep well plate
Costar
3960
BBC
M61
Reservoir Plates
Axygen
Res-SWI-LP
BBC
M62
MES
Sigma
2933
BBCf
M63
TE Buffer
Ambion
AM9858
BBC
M64
Tween-20
Boston Bioproducts
IBB-180-1L
BBC
M65
10% SDS
Ambion
AM9822
BBC
M66
50mL falcon tubes
BD Biosciences
352070
BBC
M67
1mL CryoVials
Nunc
377224
PROBE
M68
20X SSPE
M69
12X MES
M70
5M NaCl
M71
5M NaOH
M72
Luminex Magnetic Beads
M73
PCR positive control template
M74
Positive amplicon
M75
50mL conical vials
M76
Combi cassette
M77
Virus
M78
Polybrene media
M79
Puromycin
M80
CTG reagent
M81
MilliQ water
M82
TC plate, RNAi barcoded
Volume/Well
Use
Reagents internally prepared ID
Name
MIX01
MCF7 Total RNA master mix
MIX02
MIX03
RT master mix
Probe anneal master mix
Step
Composition
CAP
19.7μl TCL Lysis Buffer M01 0.3μl MCF7 Total RNA M07
20μl
Keep on ice, incubate at room temp for 5m before using
RT
3.7μl DEPC treated water M17 1μl 5X M-MLV RT Buffer M18 0.1μl 25mM dNTPs M19 0.2μl M-MLV RT enzyme M20
5μl
Keep on ice, add enzyme when ready to begin master mix addition
ANNL
4.22μl Nuclease free water M21 0.28μl "1000" probe pair mix M22 0.5μl 10X Taq ligase buffer M23
5μl
Keep at RT, warm probes before adding
MIX04
Ligation master mix
LIG
4.4375μl Nuclease free water M21 0.5μl 10X Taq ligase buffer M23 0.0625μl Taq DNA ligase M24
5μl
Keep on ice, add enzyme when ready to begin master mix addition
15μl
Keep on ice, add enzyme when ready to begin master mix addition
12.768μl Nuclease free water M21 1.5μl 10X PCR buffer M25 0.51μl MgCl2 M24 0.096μl 25mM dNTPs M19 0.015μl T3 primer 100μM M27 0.015μl T7 primer 100μM M28 0.096μl HotStarTaq M29
MIX05
PCR master mix
PCR
MIX06
SAPE master mix
SAPE
9.7μl 1X TMAC MIX08 0.3μl SAPE M37
10μl
Keep at room temperature and avoid exposure to light
HYB
M32A Bead set A (includes controls at 0.5X) M32B Bead set B (includes controls at 0.5X) MIX09 1.5X TMAC
21μl
Store at 4°C
HYB
150ml TMAC M31 1.25ml 20% Sarkosyl M33 12.5ml Tris-HCl M35 2.0ml 0.5M EDTA M36 84.25ml DDW M40
250ml
Store at room temperature
250ml
Store at room temperature
MIX07
MIX08
Coupled bead mix
1X TMAC
MIX09
1.5X TMAC
HYB
225ml TMAC M31 1.88ml 20% Sarkosyl M33 18.75ml Tris-HCl M35 3ml 0.5M EDTA M36 1.37ml DDW M40
MIX10
Bead Wash Buffer I
BBC
50μl Tween-20 M64 249.95ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
MIX11
Bead Wash Buffer II
BBC
2.5ml SDS 10% M65 247.5ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
MIX12
Wash Buffer A
HYB
300ml 20% SSPE M68 10.2ml 5M NaCl M70 699.9ml DDW M40
1000ml
Filter sterilize through 0.2μm filter, then store at room temperature
1000ml
Filter sterilize through 0.2μm filter, then store at room temperature
250ml
Bring pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C
MIX13
Wash Buffer B
HYB
83.3ml 12X MES M69 5.2ml 5M NaCl M70 0.1ml Tween-20 M64 699.9ml DDW M40
MIX14
MES Coupling Buffer
BBC
4.88g MES M62 5 drops 5M NaOH M71 249.95ml DDW M40
Overview
Protocol: Transcript Capture and Ligation Mediated Amplification In reverse transcription, mRNA transcripts from whole cell lysate or purified RNA are isolated by capturing the polyA tail of the transcript on the surface of a polyT coated turbocapture plate. Then the reverse transcriptase enzyme is used to build a cDNA complement to this polyT tail. Upstream and downstream probes containing gene specific, LUA “barcode” tags, and universal primer sequences are then added to the mix. The probes anneal and ligate to the gene specific region of the transcript. Finally, universal biotinylated primers are added to the mix and PCR amplified. The final product is biotinylated, barcoded PCR amplicon of 104-nt length.
Transcript Capture (CAP) Purpose
Immobilize mRNA from lysed cells onto the walls of the Turbocapture plate, by providing a dT coated surface which binds the poly-A tail of each mRNA transcript. RELATED PROTOCOL This step requires materials produced via the related protocol Cell Based Assay. Alternately, mRNA acquired by some other method can be used.
IMPORTANT Prepare materials and assets in pre-PCR and RNAse-free environment.
Preparation 1. 2. 3. 4.
Thaw LYSIS plates at 4°C Thaw Reference Total RNA M07 on ice Prepare CyBio tips according to plate layout for lysis transfer Label Turbocapture plates
Materials TCL buffer M01 MicroAmp seals M02 CyBio 384-well tips M03 Turbocapture plates M04 1.5ml eppendorf tubes M05
Assets Vortex A12 Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13
Reagent Mix ID
MIX01
Name MCF7 Total RNA master mix
Step
Composition
CAP
19.7μl TCL Lysis Buffer M01 0.3μl MCF7 Total RNA M07
Volume/Well
20μl
Use Keep on ice, incubate at room temp for 5m before using
Procedure 1. Label each new Turbocapture plate with information listed on the corresponding cell lysate source plate (compound set, cell line, date, plate #). Include also current date and initials. 2. Document names of plates being taken through RT on tracking sheet. 3. Centrifuge thawed cell lysate (LYSIS) plates for 1min @1000RPM. Keep plates on ice until ready to transfer. 4. Place LYSIS plate at Position 1 of CyBio and its corresponding labeled Turbocapture plate (CAP) at Position 2.
TIP: Check orientation of CAP and LYSIS plates.
5. Load CyBio 384 tip cartridge. TIP: Ensure CyBio tips have been arranged in rack to transfer only relevant lysate from LYSIS plate as specified by the ‘Plate Format’. Take care not to let any lysate get into control wells.
6. Transfer 20 μl lysate from LYSIS to CAP plate using CyBio program: 384 Lysate Capture . 7. Repeat steps 1-5 until all LYSIS plates have been transferred. 8. Prepare Reference Total RNA controls. 1. Mix Total RNA master mix MIX01 for each cell line included in ‘Plate Format’, using 1.5 ml Eppendorf tubes. Flick Eppendorf tubes to mix and incubate at room temperature for 5 min. IMPORTANT: Keep total RNA on ice, limit freeze/thaw cycles to 4, and return to -80C storage as soon as possible. Do not try to mix directly in wells.
2. Document REF RNAs prepared and their F/T# on tracking sheet. 3. Pipette 20μl of Total RNA master mix to corresponding control wells of all CAP plates according to ‘Plate Format’. 9. Seal plate with MicroAmp seal. 10. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for empty or low volume wells and mark on tracking sheet.
11. Incubate at Room Temperature for 1 hour. 12. Proceed directly to preparation for Reverse Transcription (FIRST).
Reverse Transcription (FIRST) Purpose Synthesize an immobolized cDNA that is complementary to the mRNA by using the immobilized dT as the primer and the captured mRNA as the template. IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 5x M-MLV RT buffer M18 2. Thaw 25mM dNTPs M19 3. Leave M-MLV RT Enzyme M20 at -20°C
Materials Plates from CAP step DEPC treated water M17 kept at -20° CyBio 384-well tips M03 Reagent reservoir M16
Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13 PrePCR thermocyclers A02 20° freezer, 4th floor A04
Reagent Mix ID
MIX02
Name
RT master mix
Step
RT
Composition 3.7μl DEPC treated water M17 1μl 5X M-MLV RT Buffer M18 0.1μl 25mM dNTPs M19 0.2μl M-MLV RT enzyme M20
Volume/Well
5μl
Use
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare FIRST master mix without enzyme MIX02 on ice IMPORTANT: Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out lysate from CAP plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of CAP plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. 3. Transfer 5μl of master mix from the reservoir to destination CAP plate using CyBio program: 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load CAP plate in Position 1 and place RT master mix source plate in Position 2. 3. Run CyBio program. 4. Repeat until all CAP plates have been processed. TIP: Do not let the CAP CAP plates sit dry for too long. Do not use plate stacker. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. 5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate in pre-PCR thermocycler using program: 37for90. STOP POINT: If procedure will be continued at a later time, store CAP CAP plates at -20°C, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Reverse Transcription was completed.
7. Proceed directly to preparation for Probe Anneal (ANNL).
Probe Anneal (ANNL) Purpose Bind probe pairs targeting specific sequences for genes of interest to target cDNA which corresponds to them. These sequences are located adjacent to each other on the cDNA template and contain LUA tags and universal primer sites to be used in later steps. IMPORTANT: Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 10X DNA ligase buffer M23 2. Thaw probe pool M22 at room temperature 3. Check FIRST plates for evaporated wells
Materials Plates from FIRST step CyBio 384-well tips M03 Nuclease free water M21 Reagent reservoir M16 Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Vortex A12 Plate heater A30 Benchtop centrifuge A09 CyBio Variable 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
MIX03
Name
Probe anneal master mix
Step
ANNL
Composition 4.22μl Nuclease free water M21 0.28μl "1000" probe pair mix M22 0.5μl 10X Taq ligase buffer M23
Volume/Well
5μl
Use
Keep at RT, warm probes before adding
Procedure 1. Prepare ANNL master mix without probes MIX03 at room temperature. IMPORTANT: Add probes just before beginning procedure.
TIP:Warm probes to 50 C for 10 to 20 seconds before adding.
1. Vortex to mix and pour into reagent reservoir at room temperature. 2. Spin out master mix from FIRST plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of FIRST plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 5μl of master mix from reservoir to destination FIRST plate using CyBio program 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load FIRST plate in Position 1 and load Probe Anneal master mix in Position 2. 3. Run CyBio program 4. Repeat until all FIRST plates have been processed. TIP:Do not let the FIRST plates sit dry for too long. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate overnight in Pre PCR thermocycler using program probebind6hramp.
STOP POINT: If procedure will be continued at a later time, store ANNL ANNL plates at -20°C in the 4th floor freezer A04, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Anneal was completed.
7. Proceed directly to preparation for Probe Ligation (LIG).
Probe Ligation (LIG) Purpose Ligate together the two probes of each probe pair to form a single strand comprising the entire 40 nucleotide gene specific sequence flanked by a LUA tag and T3 & T7 primer site. IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 10X Taq DNA ligase buffer M23 2. Check ANNL plates for evaporated wells 3. Note hold duration of ANNL plates on tracking sheet
Materials Plates from ANNL step CyBio 384-well tips M03 Nuclease free water M21 Taq DNA ligase enzyme M20 kept at -20°C Reagent reservoir M16 Super rags M12 Aluminum foil M13 MicroAmp seal M02
Assets Vortex A12 Plate heater A30 Benchtop centrifuge A09 CyBio Variable 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
MIX04
Name
Ligation master mix
Step
Composition
LIG
4.4375μl Nuclease free water M21 0.5μl 10X Taq ligase buffer M23 0.0625μl Taq DNA ligase M24
Volume/Well
5μl
Use
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare LIG master mix without enzyme MIX04 on ice. IMPORTANT:Keep enzyme at -20 C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out master mix from ANNL plates 1. Remove centrifuge plate holders from rotor. 2. Remove seal of ANNL plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Invert plate. 6. Place wrapped plate inverted into plate holder. 7. Spin for 1 min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 5μl of master mix from reservoir to destination ANNL plate using CyBio program 5ul Reagent Addition. 1. Load new CyBio 384 tip cartridge. TIP: Use the same tips for all transfers in this step.
2. Load ANNL plate in Position 1 and load Probe Anneal master mix in Position 2. 3. Run CyBio program 4. Repeat until all ANNL plates have been processed. TIP:Do not let the ANNL plates sit dry for too long. Additional plates can be spun out while running the CyBio.
4. Seal plates well with MicroAmp seal. IMPORTANT: Make a tight seal by pressing firmly and edging the seal with a pen.
5. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. TIP: Check for debris in wells and mark on tracking sheet.
6. Incubate overnight in Pre PCR thermocycler using program: probelig384. TIP:The program holds at 45°C for one hour, 65°C for 10 minutes, then holds overnight at 4°C.
STOP POINT: If procedure will be continued at a later time, store LIG plates at -20°C in the 4th floor freezer A04 , sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date Probe Ligation was completed.
7. Proceed directly to preparation for PCR Amplification step (PCR).
PCR Amplification (PCR) Purpose Denature ligated probe pairs from their cDNA templates, and amplify gene transcripts of interest exponentially from their cT3 and cT7 primer sites. The final products are same length, biotinylated, barcoded PCR amplicons. IMPORTANT:Prepare materials and assets in Pre-PCR and RNAse-Free environment.
Preparation 1. Thaw 25 mM dNTPs M19 with T3 M27 & T7 M38 primers 2. Thaw 25mM MgCl2 M26 3. Thaw 10X PCR buffer M25 4. Check LIG plates for evaporated wells
Materials Plates from LIG step Nuclease free water M21 HotStarTaq M29 kept at -20°C CyBio 384 well tips M03 Reagent reservoir M16 Super rag M12 Aluminum foil M13 MicroAmp seal M02
Assets Benchtop centrifuge A09 CyBio Vario 384 A01 Matrix electronic pipette A13 Pre PCR thermocycler A02 20°C freezer, 4th floor A04
Reagent Mix
ID
Name
Step
Composition
Volume/Well
Use
12.768μl Nuclease free water M21 1.5μl 10X PCR buffer M25 0.51μl MgCl2 M24 MIX05
PCR master mix
PCR
0.096μl 25mM dNTPs M19 0.015μl T3 primer 100μM M27 0.015μl T7 primer 100μM M28 0.096μl HotStarTaq M29
15μl
Keep on ice, add enzyme when ready to begin master mix addition
Procedure 1. Prepare PCR master mix without enzyme MIX05 on ice. IMPORTANT:Keep enzyme at -20°C and add just before beginning procedure. Complete as much of procedure as possible on ice.
1. Invert several times to mix and pour master mix into reagent reservoir on ice. 2. Spin out LIG plates. 1. Remove centrifuge plate holders from rotor. 2. Remove seal of LIG plate. 3. Fold Super Rag in half and place on top of plate. 4. Wrap plate in aluminum foil to reduce liquid spillage into centrifuge. 5. Place wrapped plate inverted into plate holder. TIP:See Figure 5 for configuration of plate ready to spin out.
6. Spin for 1min at 1000RPM to remove liquid from wells. TIP: Inspect plates for debris, liquid, etc. in centrifuge.
3. Transfer 15 μl of master mix from source plate to destination PCR plate using CyBio program 15 μl PCR addition. 1. Load new CyBio 384 tip cartridge TIP: Use the same tips for all transfers in this step.
2. Load LIG plate in position 1 and place PCR master mix reagent reservoir in position 2. 3. Run CyBio program 4. Repeat until all LIG plates have been processed 4. Seal the plates with MicroAmp seal IMPORTANT: Must seal plates very tightly before cycling. Press down firmly and edge wells tightly with a pen.
5. Centrifuge plates for 15 seconds at 1000RPM to settle liquid in wells
TIP:Check for debris in wells and mark on tracking sheet.
6. Incubate in 7th floor Post PCR Thermocycler using program PCR29 ALT 1M CYC. TIP: The program brings the material to 92°C, then does 29 cycles as follows: 92°C for 1 minute, 60°C for one minute, 72°C for one minute. After cycling, it holds for 5 minutes at 72°C, ad then holds at 10°C.
STOP POINT: If procedure will be continued at a later time, store PCR PCR plates at -20°C in the 7th floor freezer A05, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date PCR Amplification was completed.
7. Proceed directly to preparation for Hybridization (HYB)
Hybridization (HYB) Purpose Bind each amplified gene transcript to its corresponding Flexmap tag encoded bead in a detection plate. The tag-duo version of this protocol combines the 2 sets of 500 fluorescent beads at different proportions to create a single 1000-plex detection set instead of maintaining two separate subsets. RELATED PROTOCOL: This step requires materials produced via the related protocol Bead Preparation.
IMPORTANT: Prepare materials and assets in a Post-PCR environment.
Preparation 1. Thaw positive amplicon M74 2. Check PCR plates for evaporated wells
Materials Plates from PCR step Coupled magnetic beads set A M32A Coupled magnetic beads set B M32B 1.5X TMAC MIX09 Reagent reservoir M16 Nanoscreen tips M30 Pink microseal M34 TE buffer M63
Assets 7th floor benchtop centrifuge A10 7th floor sonicator A17 7th floor vortex A16 Biomek A27
Matrix electronic pipette A14 Post PCR thermocycler A03
Reagent Mixes ID
Name
Coupled bead mix
MIX07
MIX08
1X TMAC
MIX09
1.5X TMAC
Step
Composition
Volume/Well
Use
HYB
M32A Bead set A (includes controls at 0.5X) M32B Bead set B (includes controls at 0.5X) MIX09 1.5X TMAC
21μl
Store at 4°C
HYB
150ml TMAC M31 1.25ml 20% Sarkosyl M33 12.5ml Tris-HCl M35 2.0ml 0.5M EDTA M36 84.25ml DDW M40
250ml
Store at room temperature
HYB
225ml TMAC M31 1.88ml 20% Sarkosyl M33 18.75ml Tris-HCl M35 3ml 0.5M EDTA M36 1.37ml DDW M40
250ml
Store at room temperature
Procedure 1. Label each new HYB plate with information listed on the corresponding PCR source plate. Include also current date and initials. 2. Document names of plates being taken through hybridization on tracking sheet. 3. Prepare Coupled Bead Mix MIX07. TIP:Sonicate and vortex beads to ensure uniform dispersal.
Add 21μl bead mix into plates using Biomek program cmap prep/ bead mix addition. Reconfigure Biomek deck for PCR amplicon transfer. Run program. Add positive amplicon control 1. Dispense 5μl of positive amplicon into well B01 8. Seal plates tightly with Microseal A. 9. Incubate overnight at 45°C in 7th floor Post PCR Thermocycler using program hybridize. 4. 5. 6. 7.
TIP: This program includes a 95C denaturing step before the 45 C hold.
10. Proceed directly to preparation for SAPE Stain (SAPE).
Sape Stain (SAPE) Purpose Add a fluorescent label to each amplified gene bound to a given bead. This will allow the Luminex Flexmap3D system to quantitatively measure the amount of amplicon for each gene. IMPORTANT:Prepare materials and assets in a Post-PCR environment.
Preparation 1. Check HYB plates for evaporated wells 2. Ensure Biomek waste is empty and DDW source is full
Materials Plates from HYB step DDW M40 70% EtOH M41 1X TMAC MIX08 SAPE M37 Wash buffer A MIX12 Wash buffer B MIX13 384 well plates M38 Nanoscreen tip box cover M30
Assets 7th floor benchtop centrifuge A10 Nanoscreen A18 Biomek A27 Vortex A12 Matrix electronic pipette A14 45°C incubator A11 384 well magnets A29
Reagent Mix ID MIX06
Name SAPE master mix
Step
Composition
SAPE
9.7μl 1X TMAC MIX08 0.3μl SAPE M37
Volume/Well 10μl
Use Keep at room temperature and avoid exposure to light
Procedure TIP: Begin FlexMap 3D scanner warmup for detection step before beginning SAPE stain in order to save time.
1. Spin down HYB plates at 3000 rpm for 3 minutes. TIP: Check that beads have pelleted in the bottom of plate wells.
2. Load Biomek program file cmap cleanup that corresponds to the correct number of plates. 3. Place reagents and plates on deck as follows:
4. Run Biomek program TIP: The program will run the buffer A and B washes, and then pause for the SAPE addition.
5. Prepare SAPE master mix MIX06 6. The program will pause. Replace buffer B with reservoir of SAPE master mix and click OK to continue. TIP: Program will add 10µl SAPE mix and then pause.
7. Remove plates from Biomek deck. 8. Seal HYB plates with MicroSeal A. 9. Vortex HYB plates to mix beads and SAPE IMPORTANT: Ensure that bead pellets are fully dispersed.
10. 11. 12. 13. 14.
Incubate HYB plates in 45°C incubator for 10 minutes. Spin down plates at 3000RPM for 1 minute Remove seals and place HYB plates back on appropriate deck locations. Select OK to continue running program Seal plates with MicroSeal foil.
STOP POINT: If procedure will be continued at a later time, store SAPE SAPE plates for up to one week at 4°C in the 7th floor refrigerator A25, sealed tightly with aluminum foil or MicroAmp seals. Be sure to note the date SAPE Stain was completed.
15. Proceed directly to preparation for Detection (DET) step.
Detection (DET) Purpose Read each detection plate with the Luminex FlexMap 3D System, which will determine the identity of each bead based on the internal dye ratio, and the amount of amplicon bound to it by the fluorescence of the SAPE. IMPORTANT: Prepare materials and assets in a post PCR environment.
Materials Plates from SAPE step
Assets FlexMap 3D scanners A07
Procedure 1. Prepare FlexMap 3D scanners 1. Turn on Flexmap 3D Scanners and Computers. 2. Login to Windows 3. Login to XPonent software 4. Eject tray, and fill position RA1 with DDW, RA2 with 70% EtOH 5. Run maintenance program Daily Instrument Startup TIP: Scanner will take 30 minutes to warm up.
2. 3. 4. 5. 6. 7. 8.
Open Batch tab. Choose New Batch from Existing Protocol. Select protocol CMAP FINAL. Eject plate tray. Load SAPE plate with correct orientation Run batch. When complete, run maintenance program Daily Instrument Shutdown.
Probe Pair Preparation Probe Collapse Purpose Collapse probes from 100uM stocks into a single tube.
Preparation
1. Thaw 100μM probe stocks M54 2. Label probe aliquot tubes
Materials Cryovials M67 Reagent reservoir M16
Assets Water bath A26 Vortex A12 20°C freezer, 4th floor A04
Procedure 1. 2. 3. 4. 5.
Label probe aliquot tubes with probe pool version, date collapsed, and initials Vortex probe plates. Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. Uncap probe plates. Aspirate 2μl from each probe well using a multichannel pipette, and dispense into a reagent reservoir. TIP: Change tips each time you aspirate probes to avoid contamination.
6. 7. 8. 9. 10.
Transfer probe collapse set into a Falcon tube. Vortex collapsed probe set. Heat probes at 50C in a water bath within a beaker of distilled water for 30 seconds. Aliquot probe mix in 500μl volumes into Cryovials. Store probe aliquots in -20°C freezer.
Bead Preparation Bead Barcode Coupling (BBC) Purpose Bind the probe (and therefore target gene) specific LUA tag to each individual bead. To complete a full compiled detection, three detection plates are each coupled to different LUA tags. Calibration genes appear across all three detection plates.
Preparation 1. Prepare MES buffer MIX14 2. Thaw LUAs M56
Materials 50 ml Falcon tube M66 EDC M58 DDW M40 MES M62 Luminex magnetic microspheres M72 Collection microtubes M54
96-well deep well plate M60 Reservoir plates M61 CyBio 96 well tips M57 Reagent reservoir M16 Microseal foil M39 96 well magnets A28
Assets CyBio 96 A21 Combi A31 Sonicator A15 Vortex A12 Centrifuge A09
Reagent Mix ID
MIX14
Name
MES Coupling Buffer
Step
Composition
BBC
4.88g MES M62 5 drops 5M NaOH M71 249.95ml DDW M40
Volume/Well
250ml
Use
Bring pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C
Procedure IMPORTANT: IMPORTANT: Complete Complete this this process process as as quickly quickly as as possibly possibly to to minimize minimize bead bead exposure exposure to to light. light.
1. Aliquot 500 ml of Luminex beads in ascending order into racks of 96 deep well plates. TIP: Sonicate and vortex Luminex beads thoroughly in their stock bottles before aliquoting. Label plates with correct bead numbers.
2. Centrifuge plates at 3000 rpm for 1 minute. 3. Remove buffer from bead plates using CyBio program Remove 500 from 2 DW. 1. Place 96 well magnets on Position 1 and Position 3 of the CyBio. 2. Place 96 deep well reservoir in Position 2 to collect waste. 3. Place a bead coupling plate on each magnet and let sit one minute. TIP:The beads will pellet tightly inside the magnetic ring.
4. Load 96 well CyBio tip cartridge corresponding to the bead set. 5. Run CyBio program 4. Add MES using Combi program 65 ul, 96 DW (44 mm) Full Plate. 1. Bring bead plate to Combi. 2. Load large cassette into Combi. 3. Place Combi intake into MES buffer and prime 4. Run program 5. Add LUA tags 1. Thaw LUA plates 2. Vortex and spin down LUA plates in centrifuge (1000 rpm for 1 min).
3. Aspirate 2.5 μl of LUA from source plate. 4. Dispense 2.5 μl of LUA into its designated well in the bead plate, referring to the LUA-Bead layout map. TIP:Confirm that LUA is added to each well.
5. Repeat for all LUA-bead pairs 6. Add EDC using Combi program 65 ul, 96 DW (44 mm), Full Plate. 1. Bring all bead plates to Combi. 2. Load Small Combi cassette. 3. Mix 50 ml batch of 10 mg/mL EDC in double distilled water (DDW) and pour into reagent reservoir. 4. Run program for each plate IMPORTANT: Complete this process in 5 minutes if possible
5. 6. 7. 8.
Cover with MicroSeal foil Centrifuge plates for 15 seconds at 1000 RPM to settle liquid in wells. Incubate in dark at room temperature for 30 minutes. Repeat EDC addition and incubation for a second time with a new batch of EDC solution. Do not remove EDC from first addition. 7. Proceed directly to Bead Coupling Wash.
Bead Coupling Wash and Bead Collapse Purpose Remove all unbound LUA tags from bead mix to prevent non-specific binding of LUA tags to beads, then collapse beads into sets.
Preparation 1. Prepare Bead Wash Buffer I MIX10 2. Prepare Bead Wash Buffer II MIX11
Materials DDW M21 Tween-20 M64 SDS M65 TE buffer M63 96-well deep well plate M60 CyBio 96 well tips M57 Microseal foil M39 Reservoir plates M61 96 well bead magnet A28
Assets Vortex A12 CyBio 96 A21
Reagent Mixes
ID
Name
MIX10
Bead Wash Buffer I
MIX11
Bead Wash Buffer II
Step
Composition
Volume/Well
Use
BBC
50μl Tween-20 M64 249.95ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
BBC
2.5ml SDS 10% M65 247.5ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
Procedure 1. Wash beads with Bead Wash Buffer I using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash Buffer I and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 2. Wash beads with Bead Wash Buffer II using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash Buffer II and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 9. Vortex plate gently to disperse beads 3. Wash beads with TE Buffer using CyBio and Combi. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Bring bead plates to Combi. 6. Load large cassette into Combi. 7. Place Combi intake into Bead Wash TE and prime Combi. 8. Run Combi program 225 ul, 96 DW (44mm), Full Plate. 9. Vortex plate gently to disperse beads 4. Collapse bead set into a single tube using CyBio. 1. Place 96 well magnets on Position 1 and Position 3 of CyBio. 2. Place 96 deep well plate in Position 2 of CyBio for waste. 3. Place a bead coupling plate on top of each 96 well bead magnet and let sit 1 minute. 4. Run CyBio program Remove 250 from 2 DW. 5. Aspirate each well of the bead plate, and collapse into a 50 ml Falcon tube. 6. Dispense 50 ul of TE into the first column of the aspirated bead plate, then mix and transfer to the next column. TIP:Take a second pass through the bead plate to recover as many beads as possible.
7. Add TE with recovered beads to Falcon tube.
Assets ID
Name
Model/Vendor
Description
Unit/Location
A01
4th floor liquid handler
CyBio Vario
PERT, prePCR liquid handling, master mix additions
CyBio 384, 4th floor
A02
PrePCR thermocycler
Thermo MBS Satellite 384
FIRST - LOG, prePCR
4th floor
A03
PostPCR thermocycler
Themro MBS Satellite 384
PCR - HYB, postPCR
7th floor
A04
Freezer, -20°C
Sanyo HF-5015
FIRST-LIG, Store LMA reagents
CMAP -20°C pre, 4th floor
A05
Freezer −20°C
Sanyo MDF-U537
HYB, Store amplified PCR product
CMAP -20 post, room 7177
A06
Freezer −80°C
Sanyo MDF-U75VC
LYSIS, Store cell lysate pre-LMA
CMAP -80°C room 4051
A07
FlexMap 3D
Luminex
DET, Detect LMA product
n=9, Room 7177
A08
6 nL slot pin tool
V&P Scientific
PERT, Add small molecule
Pin tool, 4th floor
A09
Benchtop centrifuge
Eppendorf 5810
FIRST – LIG, Spin down/out mixes
prePCR, 4th floor
A10
Benchtop centrifuge
Eppendorf 5810
HYB – WASH, Spin down/out mixes
postPCR, room 7177
A11
Incubator 45°C
VWR Model 1535
SAPE, Incubate during SAPE stain
CMAP incubator, room 7177
A12
Vortex
VWR G560
CAP – ANNL, mix liquids
CMAP vortex, 4th floor
A13
prePCR multichannel
Thermo Matrix 16 Channel
CAP - PCR, add mixes to plates
CMAP prePCR matrix, 4th floor
A14
postPCR multichannel
Thermo Matrix 16 Channel
HYB - WASH, add mixes to plates
CMAP postPCR matrix, room 7177
A15
4th floor sonicator
VWR Ultrasonic, 2L
BBC, disperse settled bead
CMAP sonicator, 4th foor
A16
Vortex
VWR G560
HYB - SAPE, mix liquids
CMAP 7th floor vortex, room 7177
A17
7th floor sonicator
VWR Ultrasonic, 2L
HYB, disperse settled bead
CMAP sonicator, room 7177
A18
Liquid handler
Nanoscreen
HYB, liquid transfer postPCR
Nanoscreen 5th floor, Multimek 5th floor
A19
37°C Incubator
Thermo Scientific 3110
PLATE, cell culture incubator
CMAP TC incubator, 4th floor
A20
Plate rocker
Barnstead 4631
LYSIS, plate rocker
CMAP plate rocker, 4th floor
A21
CyBio 96
CyBio96
BBC, 96 well liquid transfer
CyBio96, 4th floor
A22
Cell counter
Beckman Coulter ViCell XR
PLATE, cell viability counter
ViCell, 4th floor TC room
A23
TC centrifuge
Beckman Coulter Allegra X-15R
PLATE, tissue culture centrifuge
TC centrifuge, 4th floor TC room
A24
TC multichannel
Ranin 1200ul pipette
PLATE, cell plating multichannel pipette
Ranin pipette, 4th floor TC room
A25
4°C fridge
SciCool SCGP210W1AREF
HYB, storage of post-hyb plates
4°C fridge, room 7177
Fisher Scientific
A26
Water bath
Isotemp 215
PROBE, prove denaturing
Water bath, 4th floor
A27
7th floor liquid handler
Biomek NX
SAPE - WASH, liquid transfer and wash postPCR
Biomek, room 7177
A28
96 well magnets
Agencourt
BBC, hold down magnetic bead
AGN#00219 | 4th floor
A29
384-well magnets
Dexter Magnetic
WASH, hold down magnetic bead
2501007 | Room 7177
A30
Plate Warmer
A31
Combi
A32
Janus liquid handler
A33
BL2+ centrifuge
A34
Electronic pipette
A35
Aspirating pipette
A36
BL2+ cell culture incubator
Reagents from external vendors ID
Name
Vendor
Catalog #
Process
M01
TCL Lysis Buffer
Invitrogen
15596-018
CAP, LYSIS
M02
MicroAmp Seal
Applied Biosystems
4306311
CAP - PCR
M03
CyBio 384 tips
CyBio
OL 3800-25-513-N
CAP - PCR
M04
384 well Turbocapture plate
Qiagen
72271
CAP - PCR
M05
1.5ml microtubes
ISC Bioexpress
C-3217-1
CAP
M06
HeLa S3 Total RNA
Ambion
Am7852
CAP
M07
MCF7 Total RNA
Ambion
AM7846
CAP
M08
HL60 Total RNA
Ambion
AM7836
CAP
M09
K562 Total RNA
Ambion
AM7832
CAP
M10
PC3 Total RNA
Ambion
AM7834
CAP
M11
MDA-MB-453 Total RNA
Ambion
AM7876
CAP
M12
Super Rag
Mercantile Development
93101
FIRST – LIG
M13
Aluminum Foil
Goldman Paper
1217
FIRST – LIG
M14
384-well plate
Eppendorf
951020737
FIRST – HYB
M15
15 ml Falcon tubes
BD Biosciences
352096
FIRST-STAIN
M16
Reagent Reservoir
Corning
4870
M17
DEPC treated water
Ambion
AM9915
FIRST
M18
5x M-MLV RT buffer
Promega
M2505
FIRST
M19
25 mM dNTPs
Invitrogen
10297018
FIRST, PCR
M20
M-MLV RT enzyme
Promega
M2505
FIRST
M21
Nuclease free water
Qiagen
XXXXX
ANNL - PCR
FIRST - STAIN
M22
Probe Dilution Plates
In-house
Custom
ANNL
M23
10x Taq ligase buffer
NEB
M0208L
ANNL, LIG
M24
Taq DNA ligase
NEB
M0208S
LIG
M25
10x PCR buffer
Qiagen
203443
PCR
M26
Magnesium Chloride
Qiagen
203443
PCR
M27
T3 primer
IDT
-
PCR
M28
T7 primer
IDT
-
PCR
M29
HotStarTaq
Qiagen
203443
PCR
M30
Nanoscreen tips 30 ul
Nanoscreen
30ulnc
HYB – WASH
M31
TMAC
Sigma
T3411-1L
HYB, WASH
M32A
Coupled Beads
In-House
Custom
HYB
M32B
Coupled Beads
In-House
Custom
HYB
M33
20% Sarkosyl
Sigma
L-9150
HYB
M34
MicroSeal
BioRad
MSA5001
HYB
M35
1M Tris-HCl, pH 8.0
Sigma
T-3038
HYB
M36
0.5M EDTA, pH 8.0
Gibco
15575-038
HYB
M37
SAPE
Invitrogen
S866
SAPE
M38
384-well plate
Costar
3456
SAPE
M39
Microseal F Foil
BioRad
MSF1001
DET
M40
Distilled Water
In House
-
DET
M41
70% Ethanol
In House
-
DET
M42
384-well cell culture plate
Corning
3570
PLATE
M43
PBS
MediaTech
21-040-CV
PLATE
M44
Trypsin EDTA
Invitrogen
25200-114
PLATE
M45
Vi-Cell Sample Vials
Beckman Coulter
383721
PLATE
M46
10cm cell culture petri dish
BD Falcon
353003
PLATE
M47
RPMI 1640 Media
Fisher Scientific
MT-10-040-CM
PLATE
M48
DMEM Media
Invitrogen
11995073
PLATE
M49
DMSO
Sigma
D2650-100ML
PERT
M50
Methanol
Fisher
A412-4
PERT
M51
1000x CAS compound plate
ChemBio
Custom
PERT
M52
Deep well 384-well plate
Costar
3965
LYSIS
M53
Luminex Carboxylated Microspheres
Luminex
-
BBC
M54
Collection Microtubes
Qiagen
19560
BBC
M55
100 µM stock probe plates
IDT
Custom
BBC
M56
LUA 96-well stock plates
IDT
Custom
BBC
M57
CyBio 96 well tips
CyBio
OL 3800-25-559-N
BBC
M58
EDC
Pierce Biosciences
22980
BBC
M59
96-well plate
Axygen
321-21-051
BBC
M60
96-well deep well plate
Costar
3960
BBC
M61
Reservoir Plates
Axygen
Res-SWI-LP
BBC
M62
MES
Sigma
2933
BBCf
M63
TE Buffer
Ambion
AM9858
BBC
M64
Tween-20
Boston Bioproducts
IBB-180-1L
BBC
M65
10% SDS
Ambion
AM9822
BBC
M66
50mL falcon tubes
BD Biosciences
352070
BBC
M67
1mL CryoVials
Nunc
377224
PROBE
M68
20X SSPE
M69
12X MES
M70
5M NaCl
M71
5M NaOH
M72
Luminex Magnetic Beads
M73
PCR positive control template
M74
Positive amplicon
M75
50mL conical vials
M76
Combi cassette
M77
Virus
M78
Polybrene media
M79
Puromycin
M80
CTG reagent
M81
MilliQ water
M82
TC plate, RNAi barcoded
Volume/Well
Use
Reagents internally prepared ID
Name
MIX01
MCF7 Total RNA master mix
MIX02
MIX03
RT master mix
Probe anneal master mix
Step
Composition
CAP
19.7μl TCL Lysis Buffer M01 0.3μl MCF7 Total RNA M07
20μl
Keep on ice, incubate at room temp for 5m before using
RT
3.7μl DEPC treated water M17 1μl 5X M-MLV RT Buffer M18 0.1μl 25mM dNTPs M19 0.2μl M-MLV RT enzyme M20
5μl
Keep on ice, add enzyme when ready to begin master mix addition
ANNL
4.22μl Nuclease free water M21 0.28μl "1000" probe pair mix M22 0.5μl 10X Taq ligase buffer M23
5μl
Keep at RT, warm probes before adding
MIX04
Ligation master mix
LIG
4.4375μl Nuclease free water M21 0.5μl 10X Taq ligase buffer M23 0.0625μl Taq DNA ligase M24
5μl
Keep on ice, add enzyme when ready to begin master mix addition
15μl
Keep on ice, add enzyme when ready to begin master mix addition
12.768μl Nuclease free water M21 1.5μl 10X PCR buffer M25 0.51μl MgCl2 M24 0.096μl 25mM dNTPs M19 0.015μl T3 primer 100μM M27 0.015μl T7 primer 100μM M28 0.096μl HotStarTaq M29
MIX05
PCR master mix
PCR
MIX06
SAPE master mix
SAPE
9.7μl 1X TMAC MIX08 0.3μl SAPE M37
10μl
Keep at room temperature and avoid exposure to light
HYB
M32A Bead set A (includes controls at 0.5X) M32B Bead set B (includes controls at 0.5X) MIX09 1.5X TMAC
21μl
Store at 4°C
HYB
150ml TMAC M31 1.25ml 20% Sarkosyl M33 12.5ml Tris-HCl M35 2.0ml 0.5M EDTA M36 84.25ml DDW M40
250ml
Store at room temperature
250ml
Store at room temperature
MIX07
MIX08
Coupled bead mix
1X TMAC
MIX09
1.5X TMAC
HYB
225ml TMAC M31 1.88ml 20% Sarkosyl M33 18.75ml Tris-HCl M35 3ml 0.5M EDTA M36 1.37ml DDW M40
MIX10
Bead Wash Buffer I
BBC
50μl Tween-20 M64 249.95ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
MIX11
Bead Wash Buffer II
BBC
2.5ml SDS 10% M65 247.5ml DDW M40
250ml
Filter sterilize through 0.2μm filter, then store at room temperature
MIX12
Wash Buffer A
HYB
300ml 20% SSPE M68 10.2ml 5M NaCl M70 699.9ml DDW M40
1000ml
Filter sterilize through 0.2μm filter, then store at room temperature
1000ml
Filter sterilize through 0.2μm filter, then store at room temperature
250ml
Bring pH to 4.5, filter sterilize through 0.2μm filter, then store at 4°C
MIX13
Wash Buffer B
HYB
83.3ml 12X MES M69 5.2ml 5M NaCl M70 0.1ml Tween-20 M64 699.9ml DDW M40
MIX14
MES Coupling Buffer
BBC
4.88g MES M62 5 drops 5M NaOH M71 249.95ml DDW M40
