Multiple Myeloma Immune Atlas Consortium: Gene Expression Profiling of the Bone Marrow Microenvironment

Preparation

Warm water bath to 37°C prior to commencing the thawing Protocol. 

Determine the cell concentration, viability using a Bio-Rad T20 Cell Counter.

Prepare ~ 42mL warm complete growth medium (e.g. 10% FBS in RPMI-1640) per sample by incubating in a 37°C water bath prior to use. 

Prepare 1X PBS with 0.04% BSA solution (keep on ice).

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) 

Add 1mLcold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an 10µL aliquot for counting.

Centrifuge cells at 370g for 0h 5m 0s at 4°C.

Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.

Using a regular-bore pipette tip, add 1mL 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).

If needed, use a cell strainer (a 40 μm Flowmi™ Tip Strainer) to remove cell debris and large clumps.

Thawing, Washing & Counting Cells (do at Room temperature) 

Remove cryovial(s) from liquid or vapor-phase nitrogen storage and immediately thaw in the water bath at 37°Cfor 0h 2m 0s. Do not submerge the entire vial in the water bath. Remove from the water bath when a tiny ice crystal remains.

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.

A	B	C	D	E	F	G
#	Sample ID	Cells per ul	Cells to capture	Vol of suspension	Vol. of Water
1
2
3
4
5
6

Appendix A

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). 

1. Centrifuge cells at 370g for 0h 5m 0s at Room temperature.
2. Remove most of the supernatant by decanting gently. Remove as much supernatant as possible. Add 100µLdead cell removal microbeads per 107 total cells. Gently resuspend cells and microbead solution using a wide-orfice pipette. Incubate for 0h 15m 0s at Room temperature.
3. During the 0h 15m 0s incubation, prepare 1X binding buffer from 20X binding buffer stock solution, e.g. dilute 500µL of 20X binding buffer stock solution with 9.5mLof 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: 

1. Place LS column in the magnetic field of a MACS separator and place a 15mL column underneath.

2. Prepare column by rinsing with 3mLof 1X binding buffer.

3. After initial 3mL has run through column, remove 15mL comical and replace with a new 15mL conical tube.

4. After cell suspension has incubated for 0h 15m 0s with microbeads, apply cell suspension to column.

5. Wash the tube holding cells with microbeads with 3mL of 1X binding buffer and apply to the column.

6. Wash column with 3mL of 1X binding buffer three more times (add subsequent wash each time the column is empty from previous wash).

7. Take resulting suspension in 15mL conical and spin down at 370g for 0h 5m 0sat 4°C. If using autoMACS column: 

8. Dilute 0.25mLof 20X binding buffer with 4.75mL of distilled water.

9. Add 500µLl of 1X binding buffer to each tube

10. Run each through DepleteS selection on the autoMACS.

11. Toss the positive fractions. Negative fractions containing the live cells should be spun down at 370g for 0h 5m 0s at 4°C.

Remove supernatant without disrupting the cell pellet. Save the removed supernatant in another tube until the protocol is complete.

Add 1mL cold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an 10µL aliquot for counting

Centrifuge cells at 370g for 0h 5m 0s at 4°C

Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.

Using a regular-bore pipette tip, add 1mL 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).

If needed, use a cell strainer (a 40 μm Flowmi™ Tip Strainer) to remove cell debris and large clumps.

Determine the cell concentration, viability using a Bio-Rad T20 Cell Counter.

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 100µLof 200,000 multiple myeloma sample cells and 100µL of 4000 NIH3T3 cells.

After thawing is complete, clean the vial with 70% alcohol and Kim Wipes.

Proceed with the 10X Genomics^® Single Cell Protocol. 

In a biosafety hood, gently transfer thawed cells to a 50mL conical tube using a wide-bore pipette tip.

Using a wide-bore pipette tip, rinse the cryovial with 1mL warm complete growth medium.

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.

Serially dilute cells with complete growth medium a total of 5 times by 1:1 volume additions with ~0h 1m 0s wait between additions (for e.g. after the first addition of 1ml, wait 0h 1m 0s, then add 2mL, wait 0h 1m 0s, then add 4 ml and so on). Add complete growth medium at a speed of 3-5 ml/sec to a total of 32mL.

Centrifuge cells at 370g for 0h 5m 0s at Room temperature.

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.

Add an additional 9mL complete growth medium (at a speed of 3-5 ml/sec) to achieve a total volume of ~10mL.

Prepare Master Mix on ice according to the standard 10X protocol and dispense 31.8µL per sample into a 8 strip tube kept on ice.  

A	B	C	D	E
Master Mix	PN	1X (μl)	4X + 10% (μl)	8X +  10% (μl)
RT Reagent B	2000165	18.8	82.2	165.0
Template Switch Oligo	3000228	2.4	10.4	20.8
Reducing Agent B	2000087	2.0	8.6	17.3
RT Enzyme C	2000085/  2000102	8.7	38.4	76.8
Total	-	31.8	139.9	279.8

Assemble Chromium Chip G in a Chromium Next GEM Secondary Holder according to the manufacturer’s instructions. 

Add 50% glycerol solution in wells that will not be used for single cell prep (70µL in row 1, 50µL in row 2, 45µL in row 3). 

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 70µL Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.

A	B	C	D	E	F	G	H	I
	Sample ID	Total counts	Live counts	Viability	Dilution	Total counts	Live counts	Viability
1
2
3
4

Appendix B

Vortex beads for 0h 0m 30s. Centrifuge the Gel Bead strip for ~0h 0m 5s. 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 50µL Gel Beads. Dispense into the bottom of wells in row labeled 2 without introducing bubbles.

Wait 0h 0m 30s and then dispense 45µL 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 (0h 17m 0s). Note any errors that occur during run.

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.

Slowly aspirate (over ~0h 0m 20s) 100µL 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 ~0h 0m 20s, dispense GEMs into the tube strip on ice with the pipette tips positioned at an angle against the sidewalls of the tubes.

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 125µL, Run Time ~0h 55m 0s

A	B	C
Step	Temperature	Time
1	53°C	00:45:00
2	85°C	00:05:00
3	4°C	Hold

Store at 4°C for up to 72h 0m 0s or at -20°C for up to a week, or proceed to the next step.  

Post GEM cleanup

Add125µLRecovery Agent to each sample at room temperature. ​DO NOT pipette mix or vortex the biphasic mixture. Wait 0h 2m 0s. The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).  

Incubate 0h 2m 0s at room temperature.  

Place on the magnet until the solution clears (~0h 5m 0s).

Transfer 35µL sample to a new tube strip. Keep on ice  

Slowly remove 125µL Recovery Agent/ Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any of the top aqueous sample.  

Prepare Dynabeads cleanup mix (DCM) according to number of reactions. Vortex well.  

A	B	C	D	E
Dynabeads Cleanup Mix              (Add reagents in the order listed)	PN	PN 1X (µl)	4X + 10% (µl)	8X + 10% (µl)
Cleanup Buffer	2000088	182	801	1602
Dynabeads MyOne SILANE Vortex thoroughly (≥30 sec) immediately before adding to the mix. If still clumpy, pipette mix to resuspend completely. DO NOT centrifuge before use.	2000048	8	35	70
Reducing Agent B	2000087	5	22	44
Nuclease-free Water		5	22	44

Add 200µL of Dynabeads Mix to each sample. Pipette mix and Incubate for 0h 10m 0s* atRoom temperature.

*At 5 minutes, pipette mix 5X. At the end of 0h 10m 0s incubation, gently place on a 10x Magnetic Separator (high end) until the solution clears (~0h 5m 0s).  

Prepare 80% ethanol. Also prepare Elution solution 1 (ES1) 

A	B	C	D
Elution Solution I  Add reagents in the order listed	PN	1X (µl)	10X (µl)
Buffer EB		98	980
10% Tween 20		1	10
Reducing Agent B	2000087	1	10
Total		100	1000

Remove the supernatant (from step 12.4). Add 300µL80% ethanol to the pellet while on the magnet. Wait 0h 0m 30s and remove the ethanol.  

Add 200µL 80% ethanol to pellet. Wait 0h 0m 30s. Remove the ethanol.  

Centrifuge briefly. Place on the magnet (low end). Remove remaining ethanol. Air dry for 0h 1m 0s.  

Remove from the magnet. Immediately add 35.5µL Elution Solution I. Pipette mix without introducing bubbles.  

cDNA Amplification

Prepare cDNA Amplification Mix on ice according to 10X recommendation. Vortex and centrifuge briefly. 

A	B	C	D	E
DNA Amplification Reaction Mix Add reagents in the order listed	PN	1X (µl)	4X + 10% (µl)	8X + 10% (µl)
Amp Mix	2000047/ 2000103	50	220	440
cDNA Primers	2000089	15	66	172
total		65	286	572

Add 65µL cDNA Amplification Reaction Mix to 35µL sample from step 12.12.  

Pipette mix and centrifuge briefly. Incubate in a thermal cycler and run the 10X  

recommended cDNA amplification program. 

A	B	C
Lid Temperature	Reaction Volume	Run Time
105°C	100 µl	~30-45 min

A	B	C
Step	Temperature	Time
1	98°C	00:03:00
2	98°C	00:00:15
3	63°C	00:00:20
4	72°C	00:01:00
5	Go to Step 2, see table below for total # of cycles
6	72°C	00:01:00
7	4°C	Hold

A	B
Cell Load	Total Cycles
˂500	13
500–6,000	12
>6,000	11

Store at 4°C for up to 72h 0m 0s or proceed to the next step.  

Vortex to resuspend the SPRIselect reagent. Add 60µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150µL). Incubate 0h 5m 0s at room temperature and place on the magnet (high end) until the solution clears (~0h 5m 0s).  

Discard the transferred supernatant without disturbing the pellet. DO NOT discard the pellet (cleanup for 3ʹ Gene Expression library construction).   

cDNA cleanup - SPRIselect 

1. Add 200µL 80% ethanol to the pellet. Wait 0h 0m 30s.
2. Remove the ethanol.
3. Repeat steps a and b for a total of 2 washes.
4. Centrifuge briefly and place on the magnet (low end).
5. Remove any remaining ethanol. Air dry for 2min. DO NOT exceed 0h 2m 0s as this will decrease elution efficiency.
6. Remove from the magnet. Add 40.5µL Buffer EB. Pipette mix 15x.
7. Incubate 0h 2m 0s at Room temperature.
8. Place the tube strip on the magnet (High end) until the solution clears.
9. Transfer 40µL sample to a new tube strip.
10. Store at 4°C for upto 72h 0m 0s or at-20°C for up to 4 weeks or proceed to 3ʹ Gene Expression Dual Index Library Construction.
11. Quantify cDNA concentration with Qubit dsDNA HS Assay Kit on the Qubit Flurometer 3. Run 1µLof sample from Pellet Cleanup from above (dilute if need to 1ng/ul) on an Agilent Bioanalyzer High Sensitivity chip to check for quality.

Fragmentation, end-repair and labeling

Prepare a thermal cycler with the 10X protocol for fragmentation. 

 

A	B	C
Lid Temperature	Reaction Volume	Run Time
65°C	50 µl	~35 min

 

A	B	C
Step	Temperature	Time
Pre-cool block Pre-cool block prior to preparing the Fragmentation Mix	4°C	Hold
Fragmentation	32°C	00:05:00
End Repair & A-tailing	65°C	00:30:00
Hold	4°C	Hold

Prepare Fragmentation Mix on ice according to instructions.  

A	B	C	D	E
Fragmentation Mix Add reagents in the order listed	PN	1X (µl)	4X + 10% (µl)	8X + 10% (µl)
Fragmentation Buffer	2000091	5	22	44
Fragmentation Enzyme	2000090/ 2000104	10	44	88
Total		15	66	132

 

Pipette mix on ice and centrifuge briefly.

Transfer ONLY 10µLpurified 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. 

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.  

Post Fragmentation, End Repair & A-tailing: ​Double Sided ​Size Selection –

SPRIselect  

Vortex to resuspend SPRIselect reagent. Add 30µL SPRIselect (0.6X) reagent to  each sample from above step. Pipette mix and incubate 0h 5m 0sat Room temperature*. 

• Prepare 80% ethanol  

Remove from the magnet. Add 50.5µL Buffer EB to each sample. Pipette mix and incubate 0h 2m 0s at Room temperature.

Place on the magnet (high end) until the solution clears and transfer 50µL sample to a new tube strip.

Place on the magnet (high end) until the solution clears and ​then transfer 75µL supernatant​ to a new tube strip.

Vortex to resuspend SPRIselect reagent. Add 10µL SPRIselect reagent (0.8X) to each sample. Pipette mix and incubate 0h 5m 0s at Room temperature.

Place on the magnet (high end) until the solution clears.

Remove80µL supernatant. DO NOT discard any beads.

Wash beads by adding 125µL 80% ethanol to the pellet. Wait0h 0m 30s.

Remove the ethanol.

Repeat steps 16.6 and 16.7 for a total of 2 washes.

Centrifuge briefly. Place on the magnet (Low end) until the solution clears. Remove remaining ethanol.

Adaptor Ligation

Prepare Adaptor Ligation Mix according to 10X protocol 

 

A	B	C	D	E
Adaptor Ligation Mix Add reagents in the order listed	PN	1X (µl)	4X + 10% (µl)	8X + 10% (µl)
Ligation Buffer	2000092	20	88	176
DNA Ligase	220110/ 220131	10	44	88
Adaptor Oligos	2000094	20	88	176
Total		50	220	440

 

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

Incubate in a thermal cycler with the 10X adaptor ligation protocol. 

 

A	B	C
Lid Temperature	Reaction Volume	Run Time
30°C	100 µl	15 min

 

A	B	C
Step	Temperature	Time
1	20°C	00:15:00
2	4°C	Hold

 

Post Ligation Cleanup with SPRIselect

Vortex to resuspend SPRIselect Reagent. Add 80µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate 0h 5m 0s at Room temperature.

Place on the magnet (high end) until the solution clears. Remove the supernatant.

To wash, add 200µL80% ethanol to the pellet. Wait0h 0m 30s.

Remove the ethanol.

Repeat steps 18.3 and 18.4 for a total of 2 washes.

Centrifuge briefly and place on the magnet (low end).

Remove any remaining ethanol. Air dry for 0h 2m 0s. DO NOT exceed 0h 2m 0s as this will decrease elution efficiency. Remove from the magnet. Add 30.5µL Buffer EB. Pipette mix and incubate 0h 2m 0s at Room temperature.

Place on the magnet (Low end) until the solution clears.

Transfer 30µL sample to a new tube strip.

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.  

Add 50µL Amp Mix (PN-2000047 or 2000103) to 30µLsample. 

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

Incubate in a thermal cycler with the following protocol. 

A	B	C
Lid Temperature	Reaction Volume	Run Time
105°C	100 µl	~25-40 min

 

A	B	C
Step	Temperature	Time
1	98°C	00:00:45
2	98°C	00:00:20
3	54°C	00:00:30
4	72°C	00:00:20
5	Go to step 2, see below for # of cycles
6	72°C	00:01:00
	4°C	Hold

 

A	B
cDNA Input	Total Cycles
1-25 ng	14-16
25-150 ng	12-14
150-500 ng	10-12
500-1,000 ng	8-10
1,000-1,500 ng	6-8

Store at 4°C up to 72h 0m 0s or proceed.

Vortex to resuspend the SPRIselect reagent. Add 60µL SPRIselect Reagent (0.6X) to each sample. Pipette mix and incubate0h 5m 0s at room temperature.

Place on the magnet until the solution clears. DO NOT discard supernatant. Transfer 150µL supernatant to a new tube strip.

Vortex to resuspend the SPRIselect reagent. Add 20µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate 0h 5m 0s atRoom temperature.

Place on the magnet until the solution clears. Then remove 165µLsupernatant. DO NOT discard any beads.

With the tube still in the magnet, add 200µL 80% ethanol to the pellet to wash the beads. Wait 0h 0m 30s.

Remove the ethanol.

Repeat steps 28 and 29 for a total of 2 washes.

Centrifuge briefly. Place on the magnet. Remove remaining ethanol.

Remove from the magnet. Add 35.5µLBuffer EB. Pipette mix and incubate 0h 2m 0s at Room temperature and then place on the magnet until the solution clears.

Transfer 35µL to a new tube strip. Store at 4°C for up to 72h 0m 0s or at -20°C for long-term storage.

Determine concentration using Qubit fluorimeter. Run 1µLsample (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.  

​3ʹ Gene Expression Library Sequencing Depth & Run Parameters  

 

Sequencing Depth: Minimum 50,000 read pairs per cell  

Sequencing Type: Paired-end, dual indexing  

   

A	B
Sequencing Read	Recommended Number of Cycles
Read 1  i7 Index  i5 Index  Read 2	28 cycles  10 cycles  10 cycles  90 cycles