Apr 18, 2025
Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) protocol
- 1La Jolla Institute for Immunology
Protocol Citation: Emil Johansson 2025. Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5n32nv1b/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 17, 2025
Last Modified: April 18, 2025
Protocol Integer ID: 130953
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000375
Abstract
This protocol has been adapted from the 10X User Guide CG000420, Rev D.
GEM Generation and Barcoding
GEM Generation and Barcoding
Prepare RT Mastermix
Prepare RT Master Mix on ice. Pipette mix 15x and centrifuge briefly
| Matermix reagent | Product number | 16X +10% (µl) | |
| RT Reagent B | 2000435/ 2000165 | 660.0 | |
| Template Switch Oligo | 3000228 | 82.7 | |
| Reducing Agent B | 2000087 | 68.6 | |
| RT Enzyme C | 2000085/ 2000436 | 308.0 |
Add 63.6 μl Master Mix into each tube of a PCR 8-tube strip on ice.
Load Chromium Next GEM Chip M
Load 130 µl 50% glycerol to row 1, 140 µl to rows 2A, 2B, 3A, and 3B.
Vortex gel beads
Mix master mix and cell suspension, and add nuclease free water to get a final volume of 105 µl.
Add 130 µl beads to each well in row 1.
Add 140 µl Master Mix + Cell Suspension to Row 2A and 2B.
Load 140 μl Partitioning Oil into rows labeled 3A and 3B.
Run on 10X Chromium Controller.
Post GEM–RT Cleanup & cDNA Amplification
Post GEM–RT Cleanup & cDNA Amplification
Transfer GEMs and perform RT
Label four tube strips and place on ice.
Press the eject button of Chromium X 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.
Visually check the volume in rows labeled 1, 2A, and 2B. Abnormally high volume relative to other wells indicates a clog. Significant volume of non-sample fluid is expected in rows 2A and 2B after a successful run and does not indicate a sample clog.
Retrieve GEMs from row labeled 3A: Slowly aspirate 90 μl GEMs from the lowest points of the recovery wells in the top row labeled 3A without creating a seal between the tips and the bottom of the wells.
Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels. Excess Partitioning Oil (clear) in the pipette tips indicates a potential clog.
Over the course of ~20 sec, dispense GEMs into first tube strip on ice with the pipette tips against the sidewalls of the tubes.
Using the same pipette tips, slowly aspirate remaining 90 μl GEMs from the wells in the top row labeled 3A and dispense in second tube strip as described above.
Retrieve from row labeled 3B: Slowly aspirate 90 μl GEMs from the lowest points of the recovery wells in the bottom row labeled 3B without creating a seal between the tips and the bottom of the wells.
10. Repeat steps 8 and 9, dispensing GEMs into third tube strip on ice with the pipette tips against the sidewalls of the tubes.
11. Using the same pipette tips, slowly aspirate remaining 90 μl GEMs from the wells in the bottom row labeled 3B and dispense in fourth tube strip as described above.
12. Transfer tube strips to a Bio-Rad T1000 thermacycler (Bio-Rad) for cDNA synthesis, performed in two steps; cDNA generation at 53°C for 45 minutes followed and reverse transcriptase deactivation at 85°C for 5 minutes.
Post GEM-RT Cleanup – Dynabeads
Add 125 μl Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min.
Slowly remove and discard 125 μl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
Prepare Dynabeads Cleanup Mix accoding to table below:
| Dynabeads Cleanup reagents | Product number | 32X +10% (µl) | |
| Cleanup Buffer | 2000088/ 2000438 | 6406.4 | |
| Dynabeads MyOne SILANE | 2000048 | 281.6 | |
| Reducing Agent B | 2000087 | 176 | |
| Nuclease-free Water | - | 176 |
Vortex and add 200 μl Dynabeads Cleanup Mix to each tube. Pipette mix 10x.
Incubate 10 min at room temperature. Pipette mix again at ~5 min after start of incubation to resuspend settled beads.
Prepare Elution Solution I according to table below:
| Elution solution reagents | Product number | 40X (µl) | |
| Buffer EB | - | 3920 | |
| 10% Tween 20 | - | 40 | |
| Reducing Agent B | 2000087 | 40 |
At the end of 10 min incubation, place the tube strips in slots 1-4 of a 10x Magnetic Separator HT (magnet High) until the solution clears.
Remove the supernatant.
Add 300 μl 80% ethanol to the pellet while on the magnet. Wait 30 sec.
Remove the ethanol.
Add 200 μl 80% ethanol to pellet. Wait 30 sec.
Remove the ethanol.
Centrifuge briefly. Place on the magnet (at Low).
Remove remaining ethanol. Air dry for 1 min.
To avoid over drying the pellet, immediately add 36 μl Elution Solution I to the tube strips, cap the tubes, remove all tube strips from the magnet, and centrifuge briefly.
10. Pipette mix (pipette set to 30 μl) without introducing bubbles.
11. Incubate 2 min at room temperature.
12. Place on the magnet (at Low) until the solution clears.
13. Transfer 35 μl sample to a new tube strip.
cDNA Amplification
Prepare cDNA Amplification Mix on ice according to table below:
| Amplification mixture reagents | Product number | 32X + 10% | |
| Amp Mix | 2000047/ 2000440 | 1760 | |
| Feature cDNA Primers 3 | 2000289 | 528 |
Add 65 μl cDNA Amplification Reaction Mix to 35 μl sample.
Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly.
Incubate in a Bio-Rad T1000 thermacycler for amplification to be performed in six steps; the enzyme was first activated at 98°C for three minutes, followed by 11 cycles where DNA was linearized at 98°C for 15 seconds, the primer annealing was performed at 63°C for 20 seconds and DNA elongation at 72°C for one minute. This was followed by a final elongation at 72°C for one minute.
cDNA Cleanup –SPRIselect
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.
Place on the magnet (at High) until the solution clears.
Transfer and save 75 μl supernatant each into two new tube strips (one for Cell Surface Protein and one for Cell Multiplexing) without disturbing the pellet. Maintain at room temperature. DO NOT discard the transferred supernatant (cleanup for Cell Surface Protein and Cell Multiplexing-step 2.3B).
Remove the remaining supernatant from the pellet without disturbing the pellet. DO NOT discard the pellet (cleanup for 3ʹ Gene Expression library construction). Immediately proceed to Pellet Cleanup.
Pellet Cleanup for 3' Gene Expression
Add 200 μl 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps i and ii for a total of 2 washes.
Centrifuge briefly and place on the magnet (at Low).
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 41 μl Buffer EB. Pipette mix 15x (pipette set to 35 μl).
Incubate 2 min at room temperature.
Place the tube strip on the magnet (at High) until the solution clears.
Transfer 40 μl sample to a new tube strip.
Transferred Supernatant Cleanup for Multiplexing
Vortex to resuspend the SPRIselect reagent. Add 70 μl SPRIselect reagent (2.0X) to 75 μl of the transferred supernatant and pipette mix 15x (pipette set to 130 μl).
Incubate for 5 min at room temperature.
Place on the magnet (at High) until the solution clears.
Remove supernatant.
Add 300 μl 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 5 and 6 for a total of 2 washes.
Centrifuge briefly and place on the magnet (at Low).
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
10. Remove from the magnet. Add 41 μl Buffer EB. Pipette mix 15x (pipette set to 35 μl).
11. Incubate 2 min at room temperature.
12. Place the tube strip on the magnet (at High) until the solution clears.
13. Transfer 40 μl sample to a new tube strip.
Post cDNA Amplification QC & Quantification
Validate fragment size by capillary electrophoresis using the Tapestation 4200 (Agilent).
3' Gene Expression Library Construction
3' Gene Expression Library Construction
Fragmentation, End Repair & A-tailing
Transfer ONLY 20 μl purified cDNA sample from Pellet Cleanup to a tube strip.
Add 15 μl Buffer EB to each sample.
Add 15 µl Fragmentation Mix to each sample.
Pipette mix 15x (pipette set to 35 μl) on ice. Centrifuge briefly.
Transfer to Bio-Rad T1000 thermacycler and incubate for 5 minutes at 32°C for cDNA fragmentation and thereafter at 65°C for 30 minutes for end repair and A-tailing to occur.
Post Fragmentation, End Repair & A-tailing Double Sided – SPRIselect
Vortex to resuspend SPRIselect reagent. Add 30 μl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 μl).
Incubate 5 min at room temperature.
Place on the magnet (at High) until the solution clears. DO NOT discard supernatant.
Transfer 75 μl supernatant to a new tube strip.
Vortex to resuspend SPRIselect reagent. Add 10 μl SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 80 μl).
Incubate 5 min at room temperature.
Place on the magnet (at High) until the solution clears.
Remove 80 μl supernatant. DO NOT discard any beads.
Add 125 μl 80% ethanol to the pellet. Wait 30 sec.
10. Remove the ethanol.
11. Repeat steps 9 and 10 for a total of 2 washes.
12. Centrifuge briefly. Place on the magnet (at Low) until the solution clears. Remove remaining ethanol pipetting slowly. DO NOT over dry to ensure maximum elution efficiency.
13. Remove from the magnet. Add 51 μl Buffer EB to each sample. Pipette mix 15x.
14. Incubate 2 min at room temperature.
15. Place on the magnet (at High) until the solution clears.
16. Transfer 50 μl sample to a new tube strip pipetting slowly.
Adaptor Ligation
Prepare adaptor ligation master mix according to table below:
| Adaptor ligation reagents | Product number | 16X + 10% | |
| Ligation Buffer | 2000092 | 352 | |
| DNA Ligase | 220110 | 176 | |
| Adaptor Oligos | 2000094 | 352 |
Add 50 μl Adaptor Ligation Mix to 50 μl sample. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly.
Incubate in a Bio-Rad T1000 thermacycler for 20 minutes at 20°C.
Post Ligation Cleanup – SPRIselect
Vortex to resuspend SPRIselect Reagent. Add 80 μl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x.
Incubate 5 min at room temperature.
Place on the magnet (at High) until the solution clears.
Remove the supernatant.
Add 200 μl 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 5 and 6 for a total of 2 washes.
Centrifuge briefly. Place on the magnet (at Low).
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
10. Remove from the magnet. Add 31 μl Buffer EB. Pipette mix 15x.
11. Incubate 2 min at room temperature.
12. Place on the magnet (at Low) until the solution clears.
13. Transfer 30 μl sample to a new tube strip.
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-3000431 Dual Index Plate TT Set A well ID) used.
Add 50 μl Amp Mix (PN-2000047) to 30 μl sample.
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.
Transfer to a Bio-Rad T1000 thermacycler and run an initial DNA denaturation and enzyme activation for 45 seconds at 98°C, followed by 5-16 cycles (depending in cDNA input),of 98°C for 15 seconds, 54°C for 30 seconds, and 72°C for 20 seconds. A final elongation at 72°C was run for one minute.
Post Sample Index PCR Double Sided Size Selection – SPRIselect
Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 μl).
Incubate 5 min at room temperature.
Place the magnet (at High) until the solution clears. DO NOT discard supernatant.
Transfer 150 μl supernatant to a new tube strip.
Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent (0.8X) to each transferred supernatant. Pipette mix 15x (pipette set to 150 μl).
Incubate 5 min at room temperature.
Place the magnet (at High) until the solution clears.
Remove 165 μl supernatant. DO NOT discard any beads.
With the tube still in the magnet, add 200 μl 80% ethanol to the pellet. Wait 30 sec.
10. Remove the ethanol.
11. Repeat steps 9 and 10 for a total of 2 washes.
12. Centrifuge briefly. Place on the magnet (at Low). Remove remaining ethanol.
13. Remove from the magnet. Add 36 μl Buffer EB. Pipette mix 15x.
14. Incubate 2 min at room temperature.
15. Place on the magnet (at Low) until the solution clears.
16. Transfer 35 μl to a new tube strip.
17. Validate size by capillary electrophoresis using the Tapestation 4200 (Agilent).
Cell Multiplexing Library Construction
Cell Multiplexing Library Construction
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-3000482 Dual Index Plate NN Set A well ID) used.
Prepare sample index PCR mixture according to table below:
| Amplification mixture reagents | Product number | 16X + 10% | |
| Amp Mix | 2000047 | 880 | |
| Buffer EB | 352 |
Transfer ONLY 10 μl DNA sample from the Transferred Supernatant Cleanup to a new tube strip.
Add 70 μl Sample Index PCR Mix to each sample.
Add 20 μl of an individual Dual Index NN Set A to each sample and record the well ID used. Pipette mix 5x (pipette set to 90 μl). Centrifuge briefly.
Incubate in a Bio-Rad T1000 thermacycler for amplification to be performed in six steps; the enzyme was first activated at 98°C for 45 seconds, followed by 6 cycles where DNA was linearized at 98°C for 20 seconds, the primer annealing was performed at 54°C for 30 seconds and DNA elongation at 72°C for 20 seconds. This was followed by a final elongation at 72°C for one minute.
Post Sample Index PCR Size Selection – SPRIselect
Vortex to resuspend the SPRIselect reagent. Add 120 μl SPRIselect Reagent (1.2X) to each sample. Pipette mix 15x (pipette set to 150 μl).
Incubate 5 min at room temperature.
Place the magnet (at High) until the solution clears.
Remove the supernatant.
Add 300 μl 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Add 200 μl 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Centrifuge briefly. Place on the magnet (at Low). Remove remaining ethanol. Air dry for 2 min.
10. Remove from the magnet. Add 41 μl Buffer EB. Pipette mix 15x.
11. Incubate 2 min at room temperature.
12. Place on the magnet (at Low) until the solution clears.
13. Transfer 40 μl to a new tube strip.
14. Validate size by capillary electrophoresis using the Tapestation 4200 (Agilent).
Sequencing
Sequencing
Sequencing was performed on a NovaSeq 6000 S4 v1.5 Flowcell (Illumina) to a depth of > 20,000 reads/cell for gene expression and > 5,000 reads/cell for hashtag antibody libraries.
Library Loading
| Instrument | Loading concentration | PhiX (%) | |
| NovaSeq | 150*/300 | 1 |
Pooling for Gene expression
| A | B | C | D | E | F | G | H | I | J | K | |
| Sample ID | # of Cells | # of Reads/Cell | Total # of Reads | Percent of Pool | nM | Target nM | IndivVol | uL FOR 5nM | uL H2O | Final Vol. | |
| HC_NR_CELSR2 Neg | 60,000 | 20,000 | 1,200,000,000 | 25 | 87.4 | 5 | 100 | 5.72 | 94.28 | 100 | |
| HC_NR_CELSR2 Pos | 60,000 | 20,000 | 1,200,000,000 | 25 | 88.3 | 5 | 100 | 5.66 | 94.34 | 100 | |
| PD_R_CELSR2 Neg | 60,000 | 20,000 | 1,200,000,000 | 25 | 93.6 | 5 | 100 | 5.34 | 94.66 | 100 | |
| PD_R_CELSR2 Pos | 60,000 | 20,000 | 1,200,000,000 | 25 | 84.9 | 5 | 100 | 5.89 | 94.11 | 100 |
Pooling for cell multiplexing
| A | B | C | D | E | F | G | H | I | J | K | |
| Sample ID | # of Cells | # of Reads/Cell | Total Reads | Percent of Pool | nM | Target nM | IndivVol | uL FOR 5nM | uL H2O | Final Vol. | |
| HC_NR_CELSR2 Neg | 60,000 | 5,000 | 300,000,000 | 25 | 144 | 5 | 100 | 3.47 | 96.53 | 100 | |
| HC_NR_CELSR2 Pos | 60,000 | 5,000 | 300,000,000 | 25 | 55.8 | 5 | 100 | 8.96 | 91.04 | 100 | |
| PD_R_CELSR2 Neg | 60,000 | 5,000 | 300,000,000 | 25 | 106 | 5 | 100 | 4.72 | 95.28 | 100 | |
| PD_R_CELSR2 Pos | 60,000 | 5,000 | 300,000,000 | 25 | 45 | 5 | 100 | 11.11 | 88.89 | 100 |
NovaSeq sequencing
Inspect the underside of each cartridge to make sure that the reservoirs are free of ice, which indicates that the reagents are thawed.
Invert each cartridge 10 times to mix reagents.
Gently tap the bottom of each cartridge on the bench to reduce air bubbles.
Without disturbing the library at the bottom, insert the uncapped library tube containing the denatured and diluted library pool into the Library Tube position (#8) of the cluster cartridge.
Insert the library tube into position #8 of the cluster cartridge.
Normalize and pool libraries and add PhiX control according to the table below:
| POOL ID | # OF SAMPLES | # OF READS/SAMPLE | TOTAL # OF READS | PERCENT OF LANE | nM | DILUTION CONC (nM) | FINAL VOLUME | uL FOR 2nM | uL RSB | FINAL VOL. | |
| Gene expression | 240,000 | 20,000 | 4.80E+09 | 50.36 | 6.5 | 2 | 156.12 | 48 | 108.11 | 156.12 | |
| Cell multiplexing | 240,000 | 5,000 | 1.20E+09 | 12.59 | 5.75 | 3 | 39.03 | 20.37 | 18.66 | 39.03 | |
| Other sample | 108,582 | 5,000 | 5.43E+08 | 5.7 | 4.54 | 2 | 17.66 | 7.78 | 9.88 | 17.66 | |
| Other sample | 108,582 | 20,000 | 2.17E+09 | 22.78 | 6.73 | 2 | 70.63 | 21 | 49.63 | 70.63 | |
| Other sample | 108,582 | 5,000 | 5.43E+08 | 5.7 | 6.12 | 3 | 17.66 | 8.66 | 9 | 17.66 | |
| Other sample | 5,932 | 5,000 | 2.97E+07 | 0.31 | 0.9 | 2 | 0.96 | 2.14 | -1.18 | 0.96 | |
| Other sample | 5,932 | 20,000 | 1.19E+08 | 1.24 | 6.98 | 2 | 3.86 | 1.11 | 2.75 | 3.86 | |
| Other sample | 5,932 | 5,000 | 2.97E+07 | 0.31 | 1.06 | 2 | 0.96 | 1.81 | -0.85 | 0.96 | |
| Phix | 1 | 95,000,000 | 9.50E+07 | 1 | 2.1 | 2 | 3.09 | 2.94 | 0.15 | 3.09 | |
| Other sample | 1 | 1,000,000 | 1.00E+06 | 0.01 | 0.03 | 2 | 0.03 | 2.51 | -2.48 | 0.03 | |
| TOTAL # OF READS: | 9.53E+09 | TOTAL VOLUME uL: | 193.68 | 310 |
Thaw the ExAmp reagents.
Prepare a fresh dilution of NaOH according to the NovaSeq 6000 Denature and Dilute Guide (document # 1000000106351).
Denature and neutralize library pool according to the NovaSeq 6000 Denature and Dilute Guide (document # 1000000106351).
Prepare the flow cell and dock
Prepare the ExAmp master mix according to table below:
| Addition order | Reagents | Volume for four-lane flow cell | |
| 1 | DPX1/JPX1 | 315 | |
| 2 | DPX2/JPX2 | 45 | |
| 3 | DPX3 | 165 |
Add 105 µl ExAmp master mix to 45 µl library pool
Load ExAmp/library mix onto the flow cell.
Load an empty library tube into position #8 of the cluster cartridge
Set Up a Sequencing Run according to table below
3' Gene Expression Library Sequencing Parameters
| A | B | |
| Parameters | Description | |
| Sequencing Depth | >20,000 read pairs per cell | |
| Sequencing Type | Paired-end, dual indexing | |
| Sequencing Read | Recommended Number of Cycles | |
| Read 1 | 28 cycles | |
| i7 Index | 10 cycles | |
| i5 Index | 10 cycles | |
| Read 2 | 90 cycles |
Cell Multiplexing Library Sequencing Parameters
| A | B | |
| Parameters | Description | |
| Sequencing Depth | >5,000 read pairs per cell | |
| Sequencing Type | Paired-end, dual indexing | |
| Sequencing Read | Recommended Number of Cycles | |
| Read 1 | 28 cycles | |
| i7 Index | 10 cycles | |
| i5 Index | 10 cycles | |
| Read 2 | 90 cycles |