TITLE: Multiple Myeloma Immune Atlas Consortium: Gene Expression Profiling of the Bone Marrow Microenvironment 
AUTHORS: Manoj Bhasin

[DESCRIPTION]
A comprehensive gene expression (GEX) profiling protocol that assists in
generating the most granular map of single cell landscape from clinical trials
supported by Multiple Myeloma Research Foundation and identify key markers of response along with opening avenues for new therapies. 

[STEPS]
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1. Preparation 
SECTION: GEM Generation and Barcoding
3. Prepare Master Mix on ice according to the standard 10X protocol and dispense 31.8 µL per sample into a 8 strip tube kept on ice.  

 


SECTION: Sample Index PCR
19. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x Sample Index name (PN-1000215 Dual Index Plate TT Set A well ID) used.  
SECTION: Post Library Construction QC
34. Determine concentration using Qubit fluorimeter. Run 1 µLsample (dilute to 1-5ng/ul) on an Agilent Bioanalyzer High Sensitivity chip.  
Determine the average fragment size from the Bioanalyzer trace. This will be used as the insert size for library quantification.  
SECTION: Sequencing the libraries
35. ​3ʹ Gene Expression Library Sequencing Depth & Run Parameters  
 
Sequencing Depth: Minimum 50,000 read pairs per cell  
Sequencing Type: Paired-end, dual indexing  
   
 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.1. Warm water bath to 37 °C prior to commencing the thawing Protocol. 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2. Thawing, Washing & Counting Cells (do at Room temperature) 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.2. Prepare ~ 42 mL warm complete growth medium (e.g. 10% FBS in RPMI-1640) per sample by incubating in a 37 °C water bath prior to use. 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.3. Prepare 1X PBS with 0.04% BSA solution (keep on ice).
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.4. Thaw a vail of NIH3T3 viably as detailed in 10X genomics protocol:  
 
CG00039_Demonstrated_Protocol_FreshFrozenHumanPBMCs_RevD.pdf 
 
Keep the thawed cells on ice till spiking into viably thawed patients’ sample (step 2.19) 
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.1. Remove cryovial(s) from liquid or vapor-phase nitrogen storage and immediately thaw in the water bath at 37 °Cfor 2 min. Do not submerge the entire vial in the water bath. Remove from the water bath when a tiny ice crystal remains.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.2. After thawing is complete, clean the vial with 70% alcohol and Kim Wipes.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.3. In a biosafety hood, gently transfer thawed cells to a 50 mL conical tube using a wide-bore pipette tip.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.4. Using a wide-bore pipette tip, rinse the cryovial with 1 mL warm complete growth medium.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.5. Using a wide-bore pipette tip, add the rinse medium dropwise (1 drop per 5 sec*) to the 50 ml conical tube while intermittently gently shaking the tube. 

*very important- add medium slowly as described.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.6. Serially dilute cells with complete growth medium a total of 5 times by 1:1 volume additions with ~1 min wait between additions (for e.g. after the first addition of 1ml, wait 1 min, then add 2 mL, wait 1 min, then add 4 ml and so on). Add complete growth medium at a speed of 3-5 ml/sec to a total of 32 mL.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.7. Centrifuge cells at 370 g for 5 min at Room temperature.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.8. Remove most of the supernatant*, resuspend cell pellet in the remaining media using a regular-bore pipette tip (or tap gently against hand palm). *Save the removed supernatant in another tube until the protocol is complete.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.9. Add an additional 9 mL complete growth medium (at a speed of 3-5 ml/sec) to achieve a total volume of ~10 mL.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.10. Determine the cell concentration using a Bio-Rad T20 Cell Counter. Calculate the total cell number (N) based on the total volume (V) and concentration (C) where N = C x V. Use the form below to note cell counts.

 
 
 
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.11. If total cell number is ≤2 x 106 cells, use the entire sample for washing. If total cell number is >2 x 106 cells, transfer ~2 million cells into a new tube for further processing.

(Note: Excess cells will be pelleted, frozen and stored for future purposes).  

Determine cell viability. All bone marrow mononuclear cell (BMMC) samples should be cleaned-up using the dead-cell removal kit prior to analysis (e.g. Miltenyi Dead Cell Removal Kit, Cat. No. 130-090-101). 

Centrifuge cells at 370 g for 5 min at Room temperature.  
Remove most of the supernatant by decanting gently. Remove as much supernatant as possible. Add 100 µLdead cell removal microbeads per 107 total cells. Gently resuspend cells and microbead solution using a wide-orfice pipette. Incubate for 15 min at Room temperature. 
During the 15 min incubation, prepare 1X binding buffer from 20X binding buffer stock solution, e.g. dilute 500 µL of 20X binding buffer stock solution with 9.5 mLof sterile, double-distilled water. Choose appropriate MACS column (LS column or autoMACS column) and MACS separator (Magnet ot autoMACS Pro Separator) for magnetic separation. 

If using LS column: 
Place LS column in the magnetic field of a MACS separator and place a 15 mL column underneath. 
Prepare column by rinsing with 3 mLof 1X binding buffer. 
After initial 3 mL has run through column, remove 15 mL comical and replace with a new 15 mL conical tube. 
After cell suspension has incubated for 15 min with microbeads, apply cell suspension to column. 
Wash the tube holding cells with microbeads with 3 mL of 1X binding buffer and apply to the column. 
Wash column with 3 mL of 1X binding buffer three more times (add subsequent wash each time the column is empty from previous wash). 
Take resulting suspension in 15 mL conical and spin down at 370 g for 5 minat 4 °C.  

If using autoMACS column: 
Dilute 0.25 mLof 20X binding buffer with 4.75 mL of distilled water. 
Add 500 µLl of 1X binding buffer to each tube 
Run each through DepleteS selection on the autoMACS. 
Toss the positive fractions. Negative fractions containing the live cells should be spun down at 370 g for 5 min at 4 °C. 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.5. Add 1 mLcold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an 10 µL aliquot for counting.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.7. Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.8. Using a regular-bore pipette tip, add 1 mL 1X PBS with 0.04% BSA or an appropriate volume to achieve a cell concentration of ~1 x 106 cells/ml. Gently pipette mix to completely suspended the cells (keep resuspended cells on ice).

(Do not invert the tube in this step, as cells can stick to the sides of the tube, thereby changing the cell concentration).
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.9. If needed, use a cell strainer (a 40 μm Flowmi™ Tip Strainer) to remove cell debris and large clumps.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.10. Determine the cell concentration, viability using a Bio-Rad T20 Cell Counter.


SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.12. Remove supernatant without disrupting the cell pellet. Save the removed supernatant in another tube until the protocol is complete.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.13. Add 1 mL cold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an 10 µL aliquot for counting 

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.15. Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.16. Using a regular-bore pipette tip, add 1 mL 1X PBS with 0.04% BSA or an appropriate volume to achieve a cell concentration of ~1 x 106 cells/ml. Gently pipette mix to completely suspended the cells (keep resuspended cells on ice).

(Do not invert the tube in this step, as cells can stick to the sides of the tube, thereby changing the cell concentration).
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.17. If needed, use a cell strainer (a 40 μm Flowmi™ Tip Strainer) to remove cell debris and large clumps.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.18. Determine the cell concentration, viability using a Bio-Rad T20 Cell Counter.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.19. Spiking in NIH3T3 cells: Adjust volume of cells to get about 2 million cells per ml. Spike in NIH3T3 cells into the patient sample at a ratio of 50:1 patient sample: murine sarcoma cells. For e.g., mix 1:1 of 100 µLof 200,000 multiple myeloma sample cells and 100 µL of 4000 NIH3T3 cells.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.20. Proceed with the 10X Genomics‱ Single Cell Protocol. 
SECTION: GEM Generation and Barcoding
4.  Assemble Chromium Chip G in a Chromium Next GEM Secondary Holder according to the manufacturer’s instructions. 
SECTION: GEM Generation and Barcoding
5. Add 50% glycerol solution in wells that will not be used for single cell prep (70 µL in row 1, 50 µL in row 2, 45 µL in row 3). 

SECTION: GEM Generation and Barcoding
6. Add appropriate volume of nuclease free water based on the cell concentration measured in step 2.19. according to ​appendix B (for targeting 5000 cells) to the master mix. Mix 4-5 times. Mix the cells and add 5000 cells (volume calculated according to the table – appendix B) to the diluted master mix. Gently mix 5X. Gently dispense 70 µL Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.

 
 
 

SECTION: GEM Generation and Barcoding
7. Vortex beads for 30 s. Centrifuge the Gel Bead strip for ~5 s. Confirm there are no bubbles at the bottom of the tubes and the liquid levels are even. Place the Gel Bead strip back in the holder. Secure the holder lid. Slowly aspirate 50 µL Gel Beads. Dispense into the bottom of wells in row labeled 2 without introducing bubbles.

SECTION: GEM Generation and Barcoding
8. Wait 30 s and then dispense 45 µL Partitioning Oil into the wells in row labeled 3. Attach 10x Gasket and load the chip on the 10X chromium controller and run the GEM generation program (Firmware V4 or higher required). Proceed to next step as soon as the run is over (17 min). Note any errors that occur during run.

SECTION: GEM Generation and Barcoding
9. Place a tube strip on ice. Press the eject button of the Controller and remove the chip. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. Ensure that the partitioning oil from the wells does not spill when exposing the wells.
SECTION: GEM Generation and Barcoding
10. Slowly aspirate (over ~20 s) 100 µL GEMs from the lowest points of the Recovery Wells in the top row without creating a seal between the tips and the bottom of the wells. Withdraw pipette tips from the wells. Check GEMs. GEMs should appear opaque and uniform across all channels. Over the course of ~20 s, dispense GEMs into the tube strip on ice with the pipette tips positioned at an angle against the sidewalls of the tubes.

SECTION: GEM Generation and Barcoding
11. Load the GEM samples from step 7 into a PCR machine and run the standard 10X recommended program for cDNA generation.

Lid Temperature 53 °C , Reaction Volume 125 µL, Run Time ~55 min


 

Store at 4 °C for up to 4320 min or at -20 °C for up to a week, or proceed to the next step.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12. Post GEM cleanup 
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.1. Add125 µLRecovery Agent to each sample at room temperature. ​DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min. The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.6. Remove the supernatant (from step 12.4). Add 300 µL80% ethanol to the pellet while on the magnet. Wait 30 s and remove the ethanol.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.5. Prepare 80% ethanol. Also prepare Elution solution 1 (ES1) 


 

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.12. Transfer 35 µL sample to a new tube strip. Keep on ice  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.11. Place on the magnet until the solution clears (~5 min). 

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.10. Incubate 2 min at room temperature.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.8. Centrifuge briefly. Place on the magnet (low end). Remove remaining ethanol. Air dry for 1 min.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.9. Remove from the magnet. Immediately add 35.5 µL Elution Solution I. Pipette mix without introducing bubbles.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13. cDNA Amplification
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13.1. Prepare cDNA Amplification Mix on ice according to 10X recommendation. Vortex and centrifuge briefly. 


 

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13.2. Add 65 µL cDNA Amplification Reaction Mix to 35 µL sample from step 12.12.  
Pipette mix and centrifuge briefly. Incubate in a thermal cycler and run the 10X  
recommended cDNA amplification program. 
 

 

 

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13.3. Store at 4 °C for up to 4320 min or proceed to the next step.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
14. cDNA cleanup - SPRIselect 

Add 200 µL 80% ethanol to the pellet. Wait 30 s.  
Remove the ethanol.  
Repeat steps a and b for a total of 2 washes.  
Centrifuge briefly and place on the magnet (low end).  
Remove any remaining ethanol. Air dry for 2min. DO NOT exceed 2 min as this will decrease elution efficiency.  
Remove from the magnet. Add 40.5 µL Buffer EB. Pipette mix 15x.  
Incubate 2 min at Room temperature.  
Place the tube strip on the magnet (High end) until the solution clears.  
Transfer 40 µL sample to a new tube strip.  
Store at 4 °C for upto 4320 min or at-20 °C for up to 4 weeks or proceed to 3ʹ Gene Expression Dual Index Library Construction.  
Quantify cDNA concentration with Qubit dsDNA HS Assay Kit on the Qubit Flurometer 3. Run 1 µLof sample from Pellet Cleanup from above (dilute if need to 1ng/ul) on an Agilent Bioanalyzer High Sensitivity chip to check for quality.  
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13.5. Discard the transferred supernatant without disturbing the pellet. DO NOT discard the pellet (cleanup for 3ʹ Gene Expression library construction).   
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
13.4. Vortex to resuspend the SPRIselect reagent. Add 60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 µL). Incubate 5 min at room temperature and place on the magnet (high end) until the solution clears (~5 min).  
SECTION: 3ʹ Gene Expression Dual Index Library Construction
15. Fragmentation, end-repair and labeling 
SECTION: 3ʹ Gene Expression Dual Index Library Construction
15.1.  Prepare a thermal cycler with the 10X protocol for fragmentation. 
 


 

 


 

SECTION: 3ʹ Gene Expression Dual Index Library Construction
15.2. Prepare Fragmentation Mix on ice according to instructions.  


 

 
Pipette mix on ice and centrifuge briefly.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
15.3. Transfer ONLY 10 µLpurified cDNA sample from from ​step 14.9​ to a tube strip. Add 25 μl Buffer EB to each sample followed by adding 15 μl Fragmentation Mix to each sample. 

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.1. Vortex to resuspend SPRIselect reagent. Add 30 µL SPRIselect (0.6X) reagent to  each sample from above step. Pipette mix and incubate 5 minat Room temperature*. 
* Prepare 80% ethanol  
SECTION: 3ʹ Gene Expression Dual Index Library Construction
15.4. Pipette mix on ice and centrifuge briefly. Transfer into the pre-cooled thermal cycler (4 °C) and press “SKIP” to initiate the protocol. After completion, proceed to next step.  

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.  Post Fragmentation, End Repair & A-tailing: ​Double Sided ​Size Selection –  
SPRIselect  
SECTION: 3ʹ Gene Expression Dual Index Library Construction
17.1. Prepare Adaptor Ligation Mix according to 10X protocol 
 


 

 
Pipette mix and centrifuge briefly. Add 50 µL Adaptor Ligation Mix to 50 µL sample from step 6.4.2.k. Pipette mix and centrifuge briefly.   

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.2. Place on the magnet (high end) until the solution clears and ​then transfer 75 µL supernatant​ to a new tube strip.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.3. Vortex to resuspend SPRIselect reagent. Add 10 µL SPRIselect reagent (0.8X) to each sample. Pipette mix and incubate 5 min at Room temperature.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.4. Place on the magnet (high end) until the solution clears.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.5. Remove80 µL supernatant. DO NOT discard any beads.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.6. Wash beads by adding 125 µL 80% ethanol to the pellet. Wait30 s.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.7. Remove the ethanol.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.8. Repeat steps 16.6 and 16.7 for a total of 2 washes.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.9. Centrifuge briefly. Place on the magnet (Low end) until the solution clears. Remove remaining ethanol.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.10. Remove from the magnet. Add 50.5 µL Buffer EB to each sample. Pipette mix and incubate 2 min at Room temperature.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
16.11. Place on the magnet (high end) until the solution clears and transfer 50 µL sample to a new tube strip.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
17. Adaptor Ligation
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18. Post Ligation Cleanup with SPRIselect
SECTION: 3ʹ Gene Expression Dual Index Library Construction
17.2. Incubate in a thermal cycler with the 10X adaptor ligation protocol. 
 


 

 


 

 
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.1. Vortex to resuspend SPRIselect Reagent. Add 80 µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate 5 min at Room temperature.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.2. Place on the magnet (high end) until the solution clears. Remove the supernatant.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.3. To wash, add 200 µL80% ethanol to the pellet. Wait30 s.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.4. Remove the ethanol.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.5. Repeat steps 18.3 and 18.4 for a total of 2 washes.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.6. Centrifuge briefly and place on the magnet (low end).
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.7. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency. Remove from the magnet. Add 30.5 µL Buffer EB. Pipette mix and incubate 2 min at Room temperature.

SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.8. Place on the magnet (Low end) until the solution clears.
SECTION: 3ʹ Gene Expression Dual Index Library Construction
18.9. Transfer 30 µL sample to a new tube strip.

SECTION: Sample Index PCR
20. Add 50 µL Amp Mix (PN-2000047 or 2000103) to 30 µLsample. 

SECTION: Sample Index PCR
21. Add 20 µL of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5x (pipette set to 90 µL). Centrifuge briefly. 

SECTION: Sample Index PCR
23. Store at 4 °C up to 4320 min or proceed.

SECTION: Sample Index PCR
22.  Incubate in a thermal cycler with the following protocol. 


 

 


 

 


 

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
24. Vortex to resuspend the SPRIselect reagent. Add 60 µL SPRIselect Reagent (0.6X) to each sample. Pipette mix and incubate5 min at room temperature.

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
25. Place on the magnet until the solution clears. DO NOT discard supernatant. Transfer 150 µL supernatant to a new tube strip.

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
26. Vortex to resuspend the SPRIselect reagent. Add 20 µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate 5 min atRoom temperature.

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
27. Place on the magnet until the solution clears. Then remove 165 µLsupernatant. DO NOT discard any beads.

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
28. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet to wash the beads. Wait 30 s.

SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
29. Remove the ethanol.
SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
30. Repeat steps 28 and 29 for a total of 2 washes.
SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
31. Centrifuge briefly. Place on the magnet. Remove remaining ethanol.
SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
32. Remove from the magnet. Add 35.5 µLBuffer EB. Pipette mix and incubate 2 min at Room temperature and then place on the magnet until the solution clears.
SECTION: Post Sample Index PCR Double Sided Size Selection with SPRIselect
33. Transfer 35 µL to a new tube strip. Store at 4 °C for up to 4320 min or at -20 °C for long-term storage.
SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
1.6.  Centrifuge cells at 370 g for 5 min at 4 °C.

SECTION: Thawing &amp; Washing bone marrow (BM) derived cells
2.14. Centrifuge cells at 370 g for 5 min at 4 °C

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.2. Slowly remove 125 µL Recovery Agent/ Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any of the top aqueous sample.  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.3. Prepare Dynabeads cleanup mix (DCM) according to number of reactions. Vortex well.  


 

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.4. Add 200 µL of Dynabeads Mix to each sample. Pipette mix and Incubate for 10 min* atRoom temperature. 

*At 5 minutes, pipette mix 5X. At the end of 10 min incubation, gently place on a 10x Magnetic Separator (high end) until the solution clears (~5 min).  

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
12.7.  Add 200 µL 80% ethanol to pellet. Wait 30 s. Remove the ethanol.