Jan 28, 2026
Methods to generate single-nucleus RNA-seq libraries from lung cancer samples
- Jixiang Zhang1,
- Sean K. Simmons1,
- Nathan Haywood1,
- Xian Adiconis1,
- Joshua Z. Levin1
- 1Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Broad-Levin
Protocol Citation: Jixiang Zhang, Sean K. Simmons, Nathan Haywood, Xian Adiconis, Joshua Z. Levin 2026. Methods to generate single-nucleus RNA-seq libraries from lung cancer samples. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpk9yvk5/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: November 05, 2025
Last Modified: January 28, 2026
Protocol Integer ID: 231590
Keywords: single-nucleus RNA-seq, lung, Nuclei isolation, chromium nuclei isolation kit cg000505, seq libraries from fresh frozen lung cancer sample, seq libraries from lung cancer sample, nucleus rna, nuclei isolation part, fresh frozen lung cancer sample, 10x genomic, lung cancer sample, cell rna, seq library, seq library part, seq platform, user guide of chromium
Funders Acknowledgements:
NIH/National Cancer Institute
Grant ID: 1U2CCA233238-01
Abstract
This protocol describes the generation of single-nucleus RNA-seq libraries from fresh frozen lung cancer samples using the 10x Genomics single-cell RNA-seq platform. It is based primarily on vendor-provided protocols with minor modifications. The nuclei isolation part is based on "User Guide for the Chromium Nuclei Isolation Kit CG000505 Rev A" (10x Genomics) and single-nucleus RNA-seq library part is based on "User guide of Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (CG000315 Rev F)"
Guidelines
Tissue collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee.
Materials
Chromium Nuclei Isolation Kit with RNase Inhibitor (#1000494, 10x Genomics)
Chromium Next GEM Chip G Single Cell Kit (#1000120, 10x Genomics)
Dual Index Kit TT Set A (#1000215, 10x Genomics)
Chromium™ Next GEM Single Cell 3' Kit v3.1 (#1000268, 10x Genomics)
Library Construction Kit (#1000190, 10x Genomics)
10x Vortex Adapter (#120251, 10x Genomics)
Chromium Next GEM Secondary Holder (#1000195, 10x Genomics)
10x Magnetic Separator (#120250, 10x Genomics)
Bio-Rad T100 Thermal Cycler
TempAssure PCR 8-tube strip (#1402-4700, USA Scientific)
SPRIselect Reagent Kit (B23318, Beckman Coulter Life Sciences)
10% Tween 20 (#1662404, Bio-Rad)
Buffer EB (#19086, Qiagen)
High Sensitivity DNA Kit (#5067-4626, Agilent)
BSA (#A2153, Sigma-Aldrich)
AOPI (#CS2-0106, Nexcelom)
CHT4 Counting Chambers (#CHT4-SD100-002, Revvity)
Koptec Pure Ethanol-200 Proof (#V1016, Decon Labs)
10x PBS (#AM9625, Ambion)
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the MSDSs (Material Safety Data Sheets).
Before start
Pre-chill centrifuge to 4 °C and place reagents and tubes on ice
Part 1: Nuclei isolation
2h 7m 23s
Pre-chill centrifuge to 4 °C and place reagents and tubes on ice as indicated in the Get Started guide. Label tops and sides of tubes, as well as tops of spin columns, before starting protocol. 80%ethanol should be prepared fresh each time.
10m
Perform all protocol steps on ice and centrifugation steps at 4 °C .
Prepare buffers according to Buffer Preparation section and place On ice .
20m
Place Sample Dissociation Tube(s) On ice
5m
Obtain frozen tissue and place immediately on dry ice.
Example of sliced lung sample as input. The tissue was frozen fresh and not fixed. The weight was around 3 mg.
Sample information
5m
Transfer frozen tissue to pre-chilled Sample Dissociation Tube.
10m
Transfer Sample Dissociation Tubes(s) to wet ice. Add 200 µL Lysis Buffer to Sample Dissociation Tube. Dissociate tissue with plastic pestle until homogeneous. For multiple samples, add Lysis Buffer to each sample and then proceed to dissociate one at a time.
Perform tissue dissociation on ice. Use one pestle per sample. DO NOT discard pestles until nuclei isolation process is complete.
10m
Add 300 µL Lysis Buffer. Pipette mix 10x. If pipette tip clogs with unhomogenized tissue, continue to dissociate tissue with the pestle until able to pipette mix.
2m
Incubate on ice for 00:10:00 .
10m
Pipette dissociated tissue into pre-chilled Nuclei Isolation Column assembled with Collection Tube using pipette set to 500 µL . Transfer all liquid from Dissociation Tube to Nuclei Isolation Column to avoid nuclei loss.
3m
Centrifuge at 16000 rcf, 4°C, 00:00:20 .
20s
Discard column. Flowthrough in the Collection Tube will contain nuclei. Vortex 00:00:10 at 3200 rpm or max speed to resuspend nuclei. Flowthrough may appear opaque or cloudy. This is normal and it is safe to proceed.
5m
Centrifuge Collection Tube for 500 rcf, 4°C, 00:03:00 . Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µL ) of supernatant if nuclei pellet is not apparent.
3m
Resuspend nuclei pellet in 500 µL Debris Removal Buffer. Gently pipette mix at least 15x, continuing until no pellet can be visualized.
2m
Centrifuge at 700 rcf, 4°C, 00:10:00 . Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µL ) of supernatant if nuclei pellet is not apparent.
10m
Resuspend nuclei pellet in 1 mL of Wash Buffer.
1m
Centrifuge at 500 rcf, 4°C, 00:05:00 . Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µL ) of supernatant if nuclei pellet is not apparent.
5m
Centrifuge at 500 rcf, 4°C, 00:05:00 . Carefully discard as much supernatant as possible using a pipette without disturbing nuclei pellet. Leave behind a small remaining volume if the pellet is not visible.
5m
Resuspend nuclei pellet in 80 µL Resuspension Buffer, depending on expected recovery for input tissue type and mass. Gently pipette mix 15x using an appropriate pipette for resuspension volume.
Resuspend in a low volume if nuclei yield is expected to be low or is unknown. DO NOT resuspend in a volume less than 50 µL .
1m
Vortex nuclei for 00:00:03 at 3200 rpm or max speed immediately prior to counting to ensure accurate nuclei count.
3s
Determine nuclei concentration using AOPI and Cellometer K2 cell counter.
20m
Part 2: GEM generation and cDNA amplification
1h 55m
Prepare Master Mix On ice . Pipette mix 15x and centrifuge briefly.
10m
Add corresponding volume of single cell suspension to Master Mix (to reach the number of 20,000 nuclei). Total of75 µL in each tube.
10m
Gently pipette mix the cell suspension before adding to the Master Mix.
2m
Gently pipette mix the Master Mix + Cell Suspension. Using the same pipette tip, dispense 70 µL Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.
2m
Load 50 µL Gel Beads, and 45 µL Partitioning Oil in row labeled 2 and 3 of Chip G, separately.
5m
Run the chip in the Chromium Controller to generate Gel Bead-in-emulsion (GEM).
30m
Transfer the GEMs into a PCR tube,
Representative GEM generation:
GEM generation
1m
Incubate in a thermal cycler with the following protocol.
Lid Temperature: 53 °C , Reaction Volume: 125 µL , Run Time: 00:55:00
Step 1: 53 °C for 00:45:00
Step 2: 85 °C for 00:05:00
Step 3: 4 °C Hold
55m
Store at 4 °C for up to 72 h or at -20 °C for up to a week or proceed to the next step.
Add 125 µL Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Wait 00:02:00 .
2m
Slowly remove and discard 125 µL Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
1m
Prepare Dynabeads Cleanup Mix.
3m
Vortex and add 200 µL to each sample. Pipette mix 10x (pipette set to 200 µL ).
2m
Incubate 00:10:00 at room temperature (keep caps open). Pipette mix again at ~00:05:00 after start of incubation to resuspend settled beads.
15m
Prepare Elution Solution I. Vortex and centrifuge briefly.
3m
At the end of 00:10:00 incubation, place on a 10x Magnetic Separator•High position (magnet•High) until the solution clears.
10m
Remove the supernatant (aqueous phase and Recovery Agent).
2m
Wash with 300 µL 80% ethanol and followed with 200 µL 80% ethanol.
Note: 80% ethanol should be prepared fresh each time.
5m
Elute with 35.5 µL Elution Solution I.
5m
Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.
5m
Add 65 µL cDNA Amplification Reaction Mix to 35 µL sample.
1m
Pipette mix 15x (pipette set to 90 µL ). Centrifuge briefly.
1m
Incubate in a thermal cycler with the following protocol (12 PCR cycles).
Lid Temperature: 105 °C , Reaction Volume: 100 µL , Run Time: ~00:45:00
Step 1: 98 °C for 00:03:00
Step 2: 98 °C for 00:00:15
Step 3: 63 °C for 00:00:20
Step 4: 72 °C for 00:01:00
Step 5: Go to Step 2, for total 12 of cycles
Step 6: 72 °C for00:01:00
Step 7: 4 °C Hold
50m 35s
Store at 4 °C for up to 72:00:00 or -20 °C for ≤1 week, or proceed to the next step.
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 ).
1m
Incubate 00:05:00 at room temperature
5m
Place on the magnet•High until the solution clears
1m
Remove the supernatant.
Add 200 µL 80% ethanol to the pellet. Wait 00:00:30 .
30s
Remove the ethanol.
Repeat steps 45.5 and 45.6 for a total of 2 washes.
3m
Centrifuge briefly and place on the magnet•Low.
Remove any remaining ethanol. Air dry for 00:02:00 . DO NOT exceed 00:02:00 as this will
decrease elution efficiency.
2m
Remove from the magnet. Add 40.5 µL Buffer EB. Pipette mix 15x (pipette set to 35 µL ).
Incubate 00:02:00 at room temperature.
2m
Place the tube strip on the magnet•High until the solution clears.
Transfer 40 µL sample to a new tube strip.
Store at 4 °C for up to 72:00:00 or at -20 °C for up to 672:00:00 , or proceed to the next step.
cDNA QC and Quantification: Run 1 µL sample on an Agilent Bioanalyzer High Sensitivity chip.
Representative cDNA traces
cDNA traces
1h
Part3: Gene Expression Dual Index Library Construction
6h 2m
Fragmentation, End Repair & A-tailing
Prepare a thermal cycler with the following incubation protocol.
Lid Temperature: 65 °C , Reaction Volume: 50 µL , Run Time: ~00:35:00
Step 1: 4 °C for Hold
Step 2: 32 °C for 00:05:00
Step 3: 65 °C for 00:30:00
Step 4: 4 °C for Hold
2m
Vortex Fragmentation Buffer. Verify there is no precipitate
2m
Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly
2m
Transfer ONLY10 µL purified cDNA sample from Pellet Cleanup to a tube
strip. Note that only 10 µL (25%) cDNA sample transfer is sufficient for
generating 3ʹ Gene Expression library. The remaining 30 µL (75%) cDNA sample
can be stored at 4 °C for up to 72 h or at 20 °C for up to 4 weeks for
generating additional 3ʹ Gene Expression libraries.
1m
Add 25 µL Buffer EB to each sample.
1m
Add 15 µL Fragmentation Mix to each sample
1m
Pipette mix 15x (pipette set to 35 µL ) on ice. Centrifuge briefly.
1m
Transfer into the pre-cooled thermal cycler (4 °C ) and press “SKIP” to initiate the protocol.
40m
Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect
40m
Vortex to resuspend SPRIselect reagent. Add 30 µL SPRIselect (0.6X) reagent to each
sample. Pipette mix 15x (pipette set to 75 µL ).
Incubate00:05:00 at room temperature
5m
Place on the magnet•High until the solution clears. DO NOT discard supernatant.
2m
Transfer 75 µL supernatant to a new tube strip.
1m
Vortex to resuspend SPRIselect reagent. Add 10 µL SPRIselect reagent (0.8X) to each transferred supernatant. Pipette mix 15x (pipette set to 80 µL ).
1m
Incubate 00:05:00 at room temperature.
5m
Place on the magnet•High until the solution clears.
2m
Remove80 µL supernatant. DO NOT discard any beads.
1m
Add 125 µL 80% ethanol to the pellet. Wait 00:00:30 .
30s
Remove the ethanol.
Repeat steps 48.9 and 48.10 for a total of 2 washes.
5m
Centrifuge briefly. Place on the magnet•Low until the solution clears. Remove
remaining ethanol. DO NOT over dry to ensure maximum elution efficiency.
1m
Remove from the magnet. Add 50.5 µL Buffer EB to each sample. Pipette mix 15x
(pipette set to 45 µL ).
1m
Incubate00:02:00 at room temperature.
2m
Place on the magnet•High until the solution clears.
2m
Transfer 50 µL sample to a new tube strip.
1m
Adaptor Ligation
20m
Prepare Adaptor Ligation Mix. Pipette mix and centrifuge briefly.
3m
Add 50 µL Adaptor Ligation Mix to 50 µL sample. Pipette mix 15x (pipette set to 90 µL ). Centrifuge briefly.
1m
Incubate in a thermal cycler with the following protocol.
Lid Temperature: 30 °C , Reaction Volume: 100 µL , Run Time: ~00:15:00
Step 1: 20 °C for 00:15:00
Step 2: 4 °C for Hold
15m
Post Ligation Cleanup – SPRIselect
20m
Vortex to resuspend SPRIselect Reagent. Add 80 µL SPRIselect Reagent (0.8X) to each
sample. Pipette mix 15x (pipette set to 150 µL ).
1m
Incubate 00:05:00 at room temperature.
5m
Place on the magnet•High until the solution clears.
2m
Remove the supernatant.
Add 200 µL 80% ethanol to the pellet. Wait 00:00:30 .
30s
Remove the ethanol.
Repeat steps 50.5 and 50.6 for a total of 2 washes.
3m
Centrifuge briefly. Place on the magnet•Low.
Remove any remaining ethanol. Air dry for 00:02:00 .
DO NOT exceed 2 min as this will decrease elution efficiency.
2m
Remove from the magnet. Add 30.5 µL Buffer EB. Pipette mix 15x.
1m
Incubate 00:02:00 at room temperature.
2m
Place on the magnet•Low until the solution clears.
1m
Transfer 30 µL sample to a new tube strip.
1m
Sample Index PCR (Dual index)
1h
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.
2m
Add 50 µL Amp Mix (PN-2000047/2000103) to 30 µL sample.
2m
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.
2m
Incubate in a thermal cycler with the following protocol .
Lid Temperature: 105 °C , Reaction Volume: 100 µL , Run Time: ~00:40:00
Step 1: 98 °C for 00:00:45
Step 2: 98 °C for 00:00:20
Step 3: 54 °C for 00:00:30
Step 4: 72 °C for00:00:20
Step 5: Go to Step 2, for total 12 of cycles
Step 6: 72 °C for 00:01:00
Step 7: 4 °C Hold
40m
Store at 4 °C for up to 72 h or at -20 °C for long-term storage.
Post Sample Index PCR Double Sided Size Selection – SPRIselect
30m
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 00:05:00 at room temperature.
5m
Place the magnet•High until the solution clears. DO NOT discard supernatant.
3m
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 ).
1m
Incubate 00:05:00 at room temperature.
5m
Place the magnet•High until the solution clears.
2m
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 00:00:30 .
30s
Remove the ethanol.
30s
Repeat steps 52.9 and 52.10 for a total of 2 washes.
5m
Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol.
1m
Remove from the magnet. Add 35.5 µL Buffer EB. Pipette mix 15x (pipette set to 35 µL ).
1m
Incubate 00:02:00 at room temperature.
2m
Place on the magnet•Low until the solution clears.
2m
Transfer 35 µL to a new tube strip.
1m
Store at 4 °C for up to 72:00:00 or at -20 °C for long-term storage.
Post Library Construction QC
1h
Run 1 µL sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.
Representative final library traces
Final library
1h
Sequencing
3d
Sequencing type: Paired end
Sequencing Depth: Minimum 20,000 read pairs per cell
Sequencing Read: Read1 28 cycles; i7 Index 10 cycles; i5 Index 10 cycles; Read 2 90 cycles.
Sequencing platform: NovaSeq X25B 300 cycles.
Example of QC data from CellRanger.
| Sample | Estimated Number of Cells | Mean Reads per Cell | Median Genes per Cell | Number of Reads | Valid Barcodes | Sequencing Saturation | Q30 Bases in Barcode | Q30 Bases in RNA Read | Q30 Bases in UMI | Reads Mapped to Genome | Reads Mapped Confidently to Genome | Reads Mapped Confidently to Intergenic Regions | Reads Mapped Confidently to Intronic Regions | Reads Mapped Confidently to Exonic Regions | Reads Mapped Confidently to Transcriptome | Reads Mapped Antisense to Gene | Fraction Reads in Cells | Total Genes Detected | Median UMI Counts per Cell | |
| 1 | 11,194 | 23,838 | 1,617 | 266,843,473 | 93.30% | 51.20% | 97.40% | 96.80% | 97.70% | 97.20% | 90.50% | 5.80% | 56.80% | 28.00% | 57.20% | 26.90% | 77.60% | 31,467 | 3,228 | |
| 2 | 12,451 | 19,063 | 1,358 | 237,358,866 | 93.50% | 52.70% | 97.00% | 96.80% | 97.50% | 97.20% | 90.70% | 5.00% | 55.30% | 30.40% | 56.70% | 28.50% | 70.00% | 30,733 | 2,288 | |
| 3 | 10,749 | 23,723 | 1,174 | 255,002,498 | 93.50% | 63.20% | 97.20% | 96.80% | 97.60% | 97.20% | 90.20% | 4.50% | 52.10% | 33.70% | 56.40% | 28.80% | 66.90% | 30,114 | 2,009 | |
| 4 | 12,121 | 21,464 | 1,877 | 260,167,621 | 94.30% | 55.00% | 97.00% | 96.80% | 97.50% | 97.10% | 89.50% | 5.20% | 57.70% | 26.60% | 65.10% | 18.40% | 84.70% | 30,799 | 3,760 | |
| 6 | 13,866 | 20,037 | 1,744 | 277,837,501 | 93.40% | 47.60% | 97.30% | 97.00% | 97.50% | 97.80% | 91.50% | 4.50% | 53.40% | 33.60% | 58.60% | 27.80% | 79.50% | 30,883 | 3,317 | |
| 7 | 8,990 | 25,878 | 1,370 | 232,641,973 | 92.10% | 53.20% | 97.20% | 96.90% | 97.60% | 96.80% | 88.60% | 5.20% | 56.70% | 26.70% | 55.80% | 27.00% | 78.60% | 30,197 | 2,462 | |
| 8 | 10,839 | 25,930 | 2,300 | 281,058,313 | 94.40% | 52.00% | 97.20% | 96.90% | 97.60% | 97.30% | 92.10% | 6.40% | 58.00% | 27.70% | 61.30% | 23.80% | 74.30% | 30,799 | 4,490 | |
| 9 | 12,505 | 22,453 | 1,506 | 280,772,363 | 94.20% | 58.30% | 97.10% | 96.60% | 97.60% | 97.00% | 90.90% | 4.90% | 54.10% | 32.00% | 60.90% | 24.50% | 70.80% | 30,946 | 2,605 |
QC from CellRanger
Example of clustering data.
Cluster data
3d
Protocol references
User Guide for the Chromium Nuclei Isolation Kit CG000505 Rev A
User guide of Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (CG000315 Rev F)
Acknowledgements
We thank Erin Kane and Sarah Mazzilli for providing sections from fresh frozen lung samples for these experiments.