May 03, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.q26g718z3gwz/v1
  • 1Emory University
Manoj Bhasin
Open access
DOI: dx.doi.org/10.17504/protocols.io.q26g718z3gwz/v1
Protocol Citation: Manoj Bhasin 2024. Multiple Myeloma Immune Atlas Consortium: Gene Expression Profiling of the Bone Marrow Microenvironment . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g718z3gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 19, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 98507
Keywords: Multiple Myeloma, scRNA-seq, Single-cell RNA-seq
Abstract
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.
Materials
S.No. Company Reagent Cat. No. 
1 10X genomics Dynabeads MyOne TM SILANE  2000048 (store at 4°C) 
2 10X genomics Chromium Next GEM Single Cell 3ʹ Gel Bead Kit v3.1, 16 rxns  1000122 (store at -20°C and -80°C according to manufacturer’s instructions) 
3 10X genomics Chromium Next Gem Chip G Single Cell Kit, 48 rxns  1000120 (store at RT) 
 4 10X genomics Dual Index Plate TT Set A 3000431 (store at −20°C) 
5 Ambion Nuclease free Water AM9937  
6 Thermo Fisher Scientific  Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)  12090-015   
7 Millipore Sigma  Ethanol, Pure (200 Proof, anhydrous)  E7023-500ML  
8 Beckman Coulter  SPRIselect Reagent Kit B23318 
9 Bio-Rad  10% Tween 20  1662404  
10 Ricca Chemical Company  Glycerin (glycerol), 50% (v/v) Aqueous Solution  3290-32  
11 Qiagen  Qiagen Buffer EB 19086  
Materials for GEM Generation and Barcoding - 1
Action  Item  10x PN  Preparation & Handling  Storage  
Equilibrate to Room Temperature  Chromium Single Cell 3’ v3 Gel Beads  2000164 Equilibrate to RT 30 min before loading the chip.  −80°C  
RT Reagent B 2000165 Vortex, verify no precipitate, centrifuge briefly.  −20°C  
Template Switch Oligo  3000228  Centrifuge briefly, resuspend in 80 μl Low TE Buffer. Vortex 15 sec at maximum speed, centrifuge briefly, leave at room temperature for ≥ 30 min. After resuspension, store at −80°C.  −80°C  
Reducing Agent B  2000087  Vortex, verify no precipitate, centrifuge briefly.  −20°C  
Place on Ice  RT Enzyme C  2000085/ 2000102  Centrifuge briefly before adding to the mix.  −20°C  
Cell Suspension  
Obtain  Partitioning Oil  2000190 -  Ambient  
Chromium Next GEM chip G  2000177  -  Ambient  
10x Gasket  370017/ 3000072   Ambient  
10x Chip Holder  330019   Ambient  
10x Vortex Adapter  330002   Ambient  
50% glycerol solution  If using <8 reactions  -   -Ambient 
Materials for GEM Generation and Barcoding -2
Action  Item  10x PN  Preparation & Handling  Storage  
Equilibrate to Room Temperature  Reducing Agent B  2000087  Thaw, vortex, verify no precipitate, centrifuge.  −20°C  
cDNA primers  2000089  Vortex, centrifuge briefly.  −20°C  
Beckman Coulter SPRIselect Reagent  -  Manufacturer’s recommendations.  -  
Agilent Bioanalyzer High Sensitivity Kit  If used for QC and quantification  -  Manufacturer’s recommendations.  -  
Agilent TapeStation ScreenTape and Reagents  If used for QC and quantification  -  Manufacturer’s recommendations.  -  
Qubit dsDNA HS Assay Kit  If used for QC and quantification  -  Manufacturer’s recommendations.  -  
Dynabeads MyOne SILANE  2000048  Vortex thoroughly (≥30 sec) immediately before adding to the mix.  4°C  
Place on ice  Amp Mix  2000047/  2000103  Vortex, centrifuge briefly.  −20°C  
Thaw at 65°C  Cleanup Buffer  2000088  Thaw for 10 min at 65°C at max speed on a thermomixer. Verify no visible crystals. Cool to room temperature.  −20°C  
Obtain  Recovery Agent  220016  -  Ambient  
Qiagen Buffer EB  -  Manufacturer’s recommendations.  -  
Bio-Rad 10% Tween 20  -  Manufacturer’s recommendations.  -  
10x Magnetic Separator  230003  -  Ambient  
Prepare 80% Ethanol - Prepare 15 ml for 8 reactions.  -  -  
Materials for Post GEM-RT Cleanup & cDNA Amplification
Obtain the following 10X kit and other components  
Action  Item  10x PN  Preparation & Handling  Storage  
Equilibrate to Room Temperature  Fragmentation Buffer  2000091  Vortex, verify no precipitate, centrifuge briefly.  −20°C  
Adaptor Oligos  2000094  Vortex, centrifuge briefly.  −20°C  
Ligation Buffer  2000092  Vortex, verify no precipitate, centrifuge briefly.  −20°C  
Dual Index Plate TT Set A  3000431 -  −20°C  
Beckman Coulter SPRIselect Reagent  B23318  Ambient 
Agilent Bioanalyzer High Sensitivity kit  If used for QC  -    
Place on Ice  Fragmentation Enzyme DNA Ligase  2000090/ 2000104  Centrifuge briefly.  −20°C 
DNA Ligase  220110 Centrifuge briefly.  −20°C  
Amp Mix 220131 Centrifuge briefly.  −20°C  
Materials for 3ʹ Gene Expression Dual Index Library Construction 
Thawing & Washing bone marrow (BM) derived cells
Thawing & Washing bone marrow (BM) derived cells
Preparation
Warm water bath to Temperature37 °C prior to commencing the thawing Protocol. 

Prepare ~ Amount42 mL warm complete growth medium (e.g. 10% FBS in RPMI-1640) per sample by incubating in a Temperature37 °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 Amount1 mL cold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an Amount10 µL aliquot for counting.

Centrifuge cells at Amount370 g for Duration00:05:00 at Temperature4 °C .

5m
Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.
Using a regular-bore pipette tip, add Amount1 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).
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.


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

Remove cryovial(s) from liquid or vapor-phase nitrogen storage and immediately thaw in the water bath at Temperature37 °C for Duration00:02:00 to 00:03:00 . Do not submerge the entire vial in the water bath. Remove from the water bath when a tiny ice crystal remains.

2m
After thawing is complete, clean the vial with 70% alcohol and Kim Wipes.
In a biosafety hood, gently transfer thawed cells to a Amount50 mL conical tube using a wide-bore pipette tip.

Using a wide-bore pipette tip, rinse the cryovial with Amount1 mL 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 ~Duration00:01:00 wait between additions (for e.g. after the first addition of 1ml, wait Duration00:01:00 , then add Amount2 mL , wait Duration00:01:00 , then add 4 ml and so on). Add complete growth medium at a speed of 3-5 ml/sec to a total of Amount32 mL .

3m
Centrifuge cells at Amount370 g for Duration00:05:00 at TemperatureRoom temperature .

5m
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 Amount9 mL complete growth medium (at a speed of 3-5 ml/sec) to achieve a total volume of ~Amount10 mL .

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.

# 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 Amount370 g for Duration00:05:00 at TemperatureRoom temperature .  
  2. Remove most of the supernatant by decanting gently. Remove as much supernatant as possible. Add Amount100 µL dead cell removal microbeads per 107 total cells. Gently resuspend cells and microbead solution using a wide-orfice pipette. Incubate for Duration00:15:00 at TemperatureRoom temperature . 
  3. During the Duration00:15:00 incubation, prepare 1X binding buffer from 20X binding buffer stock solution, e.g. dilute Amount500 µL of 20X binding buffer stock solution with Amount9.5 mL of 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 Amount15 mL column underneath. 
  2. Prepare column by rinsing with Amount3 mL of 1X binding buffer. 
  3. After initial Amount3 mL has run through column, remove Amount15 mL comical and replace with a new Amount15 mL conical tube. 
  4. After cell suspension has incubated for Duration00:15:00 with microbeads, apply cell suspension to column. 
  5. Wash the tube holding cells with microbeads with Amount3 mL of 1X binding buffer and apply to the column. 
  6. Wash column with Amount3 mL of 1X binding buffer three more times (add subsequent wash each time the column is empty from previous wash). 
  7. Take resulting suspension in Amount15 mL conical and spin down at Amount370 g for Duration00:05:00 at Temperature4 °C .  
If using autoMACS column: 
  1. Dilute Amount0.25 mL of 20X binding buffer with Amount4.75 mL of distilled water. 
  2. Add Amount500 µL l of 1X binding buffer to each tube 
  3. Run each through DepleteS selection on the autoMACS. 
  4. Toss the positive fractions. Negative fractions containing the live cells should be spun down at Amount370 g for Duration00:05:00 at Temperature4 °C . 

1h
Remove supernatant without disrupting the cell pellet. Save the removed supernatant in another tube until the protocol is complete.
Add Amount1 mL cold 1X PBS with 0.04% BSA (40 mg/100ml) and gently pipette mix 5 times with a wide bore tip. Take an Amount10 µL aliquot for counting

Centrifuge cells at Amount370 g for Duration00:05:00 at Temperature4 °C

5m
Remove supernatant without disrupting the cell pellet. Save the removed until the Protocol is complete.
Using a regular-bore pipette tip, add Amount1 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).
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 Amount100 µL of 200,000 multiple myeloma sample cells and Amount100 µL of 4000 NIH3T3 cells.

Proceed with the 10X Genomics‱ Single Cell Protocol. 
GEM Generation and Barcoding
GEM Generation and Barcoding
Prepare Master Mix on ice according to the standard 10X protocol and dispense Amount31.8 µL per sample into a 8 strip tube kept on ice.  
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 (Amount70 µL in row 1, Amount50 µL in row 2, Amount45 µL 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 Amount70 µL Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.

   Sample ID Total counts Live counts Viability Dilution  Total counts Live counts Viability 
1         
2         
3         
4         
         
         
         
Appendix B

Vortex beads for Duration00:00:30 . Centrifuge the Gel Bead strip for ~Duration00:00:05 . 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 Amount50 µL Gel Beads. Dispense into the bottom of wells in row labeled 2 without introducing bubbles.

35s
Wait Duration00:00:30 and then dispense Amount45 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 (Duration00:17:00 ). Note any errors that occur during run.

17m 30s
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 ~Duration00:00:20 ) Amount100 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 ~Duration00:00:20 , dispense GEMs into the tube strip on ice with the pipette tips positioned at an angle against the sidewalls of the tubes.

40s
Load the GEM samples from step 7 into a PCR machine and run the standard 10X recommended program for cDNA generation.

Lid Temperature Temperature53 °C , Reaction Volume Amount125 µL , Run Time ~Duration00:55:00
Step Temperature Time 
1 53°C 00:45:00 
2 85°C 00:05:00 
3 4°C Hold 

Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for up to a week, or proceed to the next step.  

3d 0h 55m
Post GEM-RT Cleanup & cDNA Amplification
Post GEM-RT Cleanup & cDNA Amplification
Post GEM cleanup
AddAmount125 µL Recovery Agent to each sample at room temperature. ​DO NOT pipette mix or vortex the biphasic mixture. Wait Duration00:02:00 . The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).  

2m
Slowly remove Amount125 µL 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.  
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 Amount200 µL of Dynabeads Mix to each sample. Pipette mix and Incubate for Duration00:10:00 * atTemperatureRoom temperature .

*At 5 minutes, pipette mix 5X. At the end of Duration00:10:00 incubation, gently place on a 10x Magnetic Separator (high end) until the solution clears (~Duration00:05:00 ).  

25m
Prepare 80% ethanol. Also prepare Elution solution 1 (ES1) 
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 Amount300 µL 80% ethanol to the pellet while on the magnet. Wait Duration00:00:30 and remove the ethanol.  

30s
Add Amount200 µL 80% ethanol to pellet. Wait Duration00:00:30 . Remove the ethanol.  

30s
Centrifuge briefly. Place on the magnet (low end). Remove remaining ethanol. Air dry for Duration00:01:00 .  

1m
Remove from the magnet. Immediately add Amount35.5 µL Elution Solution I. Pipette mix without introducing bubbles.  

Incubate Duration00:02:00 at room temperature.  

2m
Place on the magnet until the solution clears (~Duration00:05:00 ).

5m
Transfer Amount35 µL sample to a new tube strip. Keep on ice  

cDNA Amplification
Prepare cDNA Amplification Mix on ice according to 10X recommendation. Vortex and centrifuge briefly. 
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 Amount65 µL cDNA Amplification Reaction Mix to Amount35 µL sample from step 12.12.  
Pipette mix and centrifuge briefly. Incubate in a thermal cycler and run the 10X  
recommended cDNA amplification program. 
Lid Temperature Reaction Volume  Run Time 
105°C  100 µl  ~30-45 min 

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 

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

Store at Temperature4 °C for up to Duration72:00:00 or proceed to the next step.  

3d
Vortex to resuspend the SPRIselect reagent. Add Amount60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to Amount150 µL ). Incubate Duration00:05:00 at room temperature and place on the magnet (high end) until the solution clears (~Duration00:05:00 ).  
10m
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 Amount200 µL 80% ethanol to the pellet. Wait Duration00:00:30 .  
  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 Duration00:02:00 as this will decrease elution efficiency.  
  6. Remove from the magnet. Add Amount40.5 µL Buffer EB. Pipette mix 15x.  
  7. Incubate Duration00:02:00 at TemperatureRoom temperature .  
  8. Place the tube strip on the magnet (High end) until the solution clears.  
  9. Transfer Amount40 µL sample to a new tube strip.  
  10. Store at Temperature4 °C for upto Duration72:00:00 or atTemperature-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 Amount1 µL of sample from Pellet Cleanup from above (dilute if need to 1ng/ul) on an Agilent Bioanalyzer High Sensitivity chip to check for quality.  
3d 0h 4m 30s
3ʹ Gene Expression Dual Index Library Construction
3ʹ Gene Expression Dual Index Library Construction
Fragmentation, end-repair and labeling 
Prepare a thermal cycler with the 10X protocol for fragmentation. 
 
Lid Temperature Reaction Volume Run Time 
65°C 50 µl ~35 min 

 
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.  
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 Amount10 µL purified 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 (Temperature4 °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 Amount30 µL SPRIselect (0.6X) reagent to  each sample from above step. Pipette mix and incubate Duration00:05:00 at TemperatureRoom temperature *. 
* Prepare 80% ethanol  
5m
Place on the magnet (high end) until the solution clears and ​then transfer Amount75 µL supernatant​ to a new tube strip.

Vortex to resuspend SPRIselect reagent. Add Amount10 µL SPRIselect reagent (0.8X) to each sample. Pipette mix and incubate Duration00:05:00 at TemperatureRoom temperature .

5m
Place on the magnet (high end) until the solution clears.
RemoveAmount80 µL supernatant. DO NOT discard any beads.

Wash beads by adding Amount125 µL 80% ethanol to the pellet. WaitDuration00:00:30 .

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.
Remove from the magnet. Add Amount50.5 µL Buffer EB to each sample. Pipette mix and incubate Duration00:02:00 at TemperatureRoom temperature .

2m
Place on the magnet (high end) until the solution clears and transfer Amount50 µL sample to a new tube strip.

Adaptor Ligation
Prepare Adaptor Ligation Mix according to 10X protocol 
 
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 Amount50 µL Adaptor Ligation Mix to Amount50 µL sample from step 6.4.2.k. Pipette mix and centrifuge briefly.   

Incubate in a thermal cycler with the 10X adaptor ligation protocol. 
 
Lid Temperature Reaction Volume Run Time 
30°C  100 µl  15 min 

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

 
Post Ligation Cleanup with SPRIselect
Vortex to resuspend SPRIselect Reagent. Add Amount80 µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate Duration00:05:00 at TemperatureRoom temperature .

5m
Place on the magnet (high end) until the solution clears. Remove the supernatant.
To wash, add Amount200 µL 80% ethanol to the pellet. WaitDuration00:00:30 .

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 Duration00:02:00 . DO NOT exceed Duration00:02:00 as this will decrease elution efficiency. Remove from the magnet. Add Amount30.5 µL Buffer EB. Pipette mix and incubate Duration00:02:00 at TemperatureRoom temperature .

6m
Place on the magnet (Low end) until the solution clears.
Transfer Amount30 µL sample to a new tube strip.

Sample Index PCR
Sample Index PCR
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 Amount50 µL Amp Mix (PN-2000047 or 2000103) to Amount30 µL sample. 

Add Amount20 µL of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5x (pipette set to Amount90 µL ). Centrifuge briefly. 

Incubate in a thermal cycler with the following protocol. 


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

 
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 

 
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 Temperature4 °C up to Duration72:00:00 or proceed.

3d
Post Sample Index PCR Double Sided Size Selection with SPRIselect
Post Sample Index PCR Double Sided Size Selection with SPRIselect
Vortex to resuspend the SPRIselect reagent. Add Amount60 µL SPRIselect Reagent (0.6X) to each sample. Pipette mix and incubateDuration00:05:00 at room temperature.

5m
Place on the magnet until the solution clears. DO NOT discard supernatant. Transfer Amount150 µL supernatant to a new tube strip.

Vortex to resuspend the SPRIselect reagent. Add Amount20 µL SPRIselect Reagent (0.8X) to each sample. Pipette mix and incubate Duration00:05:00 atTemperatureRoom temperature .

5m
Place on the magnet until the solution clears. Then remove Amount165 µL supernatant. DO NOT discard any beads.

With the tube still in the magnet, add Amount200 µL 80% ethanol to the pellet to wash the beads. Wait Duration00:00:30 .

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 Amount35.5 µL Buffer EB. Pipette mix and incubate Duration00:02:00 at TemperatureRoom temperature and then place on the magnet until the solution clears.
2m
Transfer Amount35 µL to a new tube strip. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for long-term storage.
3d
Post Library Construction QC
Post Library Construction QC
Determine concentration using Qubit fluorimeter. Run Amount1 µL sample (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.  
Sequencing the libraries
Sequencing the libraries
​3ʹ Gene Expression Library Sequencing Depth & Run Parameters  
 
Sequencing Depth: Minimum 50,000 read pairs per cell  
Sequencing Type: Paired-end, dual indexing  
   
Sequencing Read  Recommended Number of Cycles  
Read 1  i7 Index  i5 Index  Read 2  28 cycles  10 cycles  10 cycles  90 cycles  

Protocol references