Apr 16, 2026

GIFT-seq: Nuclei Preparation

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5kwkdv1b/v1
  • 1Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY;
  • 2INSERM UMR 1342, Université Paris Cité, Assistance publique des Hôpitaux de Paris (APHP), Hôpital Saint-Louis, Paris, France
Karolis Kumpaitis
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5kwkdv1b/v1
Protocol Citation: Karolis Kumpaitis, Nabih Maslah, Catherine Snopkowski, Sydney B. Blattman, Austin A. Varela, Ronan Chaligné, Caleb Lareau 2026. GIFT-seq: Nuclei Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5kwkdv1b/v1
Manuscript citation:
Blattman, S. B. et al. Scalable genotyping in fixed transcriptomes resolves clonal heterogeneity via single-cell sequencing. bioRxiv (2026) doi: https://doi.org/10.64898/2026.04.11.717967
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: December 11, 2025
Last Modified: April 16, 2026
Protocol  Integer ID: 234829
Keywords: FFPE, GIFT-seq, Gapfill, Fixation, Nuclei, Nuclei preparation, Nuclei isolation, Decrosslinking, 10x Genomics, Single-cell, Flex, Flex, JAK2, nuclei preparation this protocol, nuclei preparation, tissue derived nuclei, nuclei for gem, 10x genomic, derived nuclei, nuclei, preparation of formalin, fixation of cell, library preparation protocol, seq, ffpe, preparation, gapfill, tissue
Abstract
This protocol can be used for preparation of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Derived Nuclei. Together should be used - 10x Genomics "Fixation of Cells & Nuclei for GEM-X Flex Gene Expression" (CG000782 | Rev D). This protocol should be followed by "GIFT-seq: Gapfill and Library Preparation Protocol".
Guidelines
  • Maintain an RNAse free environment when preparing buffers and throughout the protocol by spraying bench with RNaseZap™ and using molecular grade reagents when possible.
  • To increase cell recovery, opt for a swinging bucket rotor when pelleting and leave a 10-30 µL of liquid behind when aspirating buffers.
  • When possible, use low-binding plasticware.
Materials
  1. Sorting & Collection (S&C) Buffer: 1x PBS (Corning cat# 21-040-CV) + 0.02% BSA (Gibco cat# 15260037) + 0.2 U/uL RNase Inhibitor (Sigma Aldrich cat# 3335402001)
  2. Long-term Storage Buffer: Water (Invitrogen cat# 10977-015) + 1x Quenching Buffer B (10x Genomics PN-2001300) + 10% Glycerol (Millipore Sigma cat# G5516-100ML) + 0.1 Enhancer (10x Genomics PN-2000482) (warmed for 10 minutes at 65°C 1000 rpm)
  3. Additive C (10x Genomics PN-2001332)
  4. PBS-T: 1x PBS (Corning cat# 21-040-CV) + 0.1% Tween20 (10%) (Bio Rad cat# 1610781)
  5. Formaldehyde (37 %) (Fisher Scientific cat# BP531-25)
  6. Glycerin (Glycerol) (50 % (v/v)) (Ricca Chemical Company cat# 3290-32)
  7. SYTO 9 Green Fluorescent Nucleic Acid Stain (Invitrogen cat# S34854)
  8. DAPI Nucleic Acid Stain (1 mg/mL) (Thermo Scientific cat# 62248)
  9. Urea (8M) (Sigma-Aldrich cat# 51457-100ML)
  10. Proteinase K working solution (1:500): 1x PBS + 40 µg/mL Proteinase K (New England Biolabs cat# P8107S)
  11. 2-amino-5-methylbenzoic acid (2A5MBA) powder (Sigma-Aldrich cat# 419443-5G)
  • 1 g 2A5MBA in PBS Final volume - 40.8 mL
  • Mix 1g in ~20 mL RNAse/DNAse free Water + 4.08 mL 10x PBS (Corning cat# 46-013-CM)
  • Progressively add 1 N NaOH (Millipore Sigma cat# S2770-100ML) to dissolve, should start dissolving around 5.5 mL 1 N NaOH and slowly add more. Check pH with pH paper once all powder is dissolved, and add NaOH until pH ~7.4. May have to vortex and moderately heat to 37°C.
  • Top up to 40.8 mL with water. Filter, aliquot for single-use 500 µL and freeze. Not tested for freeze and thaw cycles.
Protocol materials
Protector RNase inhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Safety warnings
Use formaldehyde with caution as it is a hazardous material.
Ethics statement
Our research complies with all relevant ethical and regulatory guidance. Recruitment of patients was approved
under IDRCB 2023-A01414-41. Patient samples analyzed in this study were fully de-identified prior to transfer
and analysis. The use of these specimens for the present work was reviewed and determined to be exempt
from human subjects research by the Institutional Review Board of Memorial Sloan Kettering Cancer Center.
Nuclei Preparation from Formalin Fixed Paraffin Embedded (FFPE) Tissue (With S2 Genomics Singulator)
1h 14m
NUCLEI ISOLATION
Obtain 2 to 6 FFPE curls each 50 µm to 80 µm of thickness.
Curls can be stored in a 1.5mL tube at 4 °C for long term storage.
Note
Number of curls and thickness will change based on how much tissue is embedded and what is desired or needed number of nuclei.

Preparation:
  • For each sample, pre-cool 2 NIC+ (yellow) cartridges (S2 Genomics) at 4 °C orOn ice .
  • Turn on Singulator and tablet. Start FFPE protocol and let machine preheat 00:10:00 approx. .
Equipment
Singulator 100 System
NAME
Tissue dissociation
TYPE
S2 Genomics
BRAND
100-067-764
SKU
Note. If pop-up appears saying that temperature isn't reached, press "Wait".
10m
Take 1 NIC+ Cartridge, remove grinder from the cartridge and add curls to the compartment. Put grinder head back.

Manually press and spin grinder head a few times to make sure curls are flat on the bottom of the cartridge chamber.
Once S100 reaches temperature, insert NIC+ Cartridge with flattened out curl.

Start run. Deparaffination and rehydration takes 00:28:00 .
28m
During the run, pre-cool Singulator 200 for subsequent nuclei extraction from rehydrated tissue.
Upon completion of the protocol, remove the cartridge from the machine.
Equipment
Singulator 200 System
NAME
Tissue dissociation
TYPE
S2 Genomics
BRAND



Transfer tissue to cold NIC+ cartridge one of the following ways:
  • Transfer tissue pieces using forceps to NIC+ cartridge for nuclei isolation and add 500 µL of nuclei isolation reagent (NIR; S2 Genomics) and 62.5 µL RNase Inhibitor
  • Using serological pipette remove sample (will be in pieces) from the dissociation chamber and place in 5 mL Protein LoBind tube:
  1. Centrifuge1000 x g, 4°C, 00:03:00
  2. Remove supernatant and resuspend in 500 µL of NIR and add 62.5 µL RNase Inhibitor
  3. Add tissue in NIR to cold NIC+ cartridge
Protector RNase inhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001

Note
Rnase Inhibitor can be added based on tissue RNase activity. Lowest suggested final concentration is 0.2 U/µL (12.5 µL) and up to 1 U/µL (62.5 µL) as maximum suggested final concentration.

3m
Place cartridge in Singulator 200 and press start. Protocol takes 00:18:00 .

18m
While Singulator 200 is running prepare 1 mL of nuclei storage reagent (NSR; S2 Genomics) with 1 U/µL RNase inhibitor by adding 25 µL to 1 mL of NSR.
Upon completion of the run, remove cartridge and transfer sample (About 2 mL) into 5 mL Protein LoBind tube.
Centrifuge 500 x g, 4°C, 00:05:00 .
5m
Remove supernatant and resuspend in 1 mL NSR with RNase inhibitor.
Filter nuclei suspension with 30 µm filter to new 1.5 mL Protein LoBind tube.
Equipment
Pre-Separation Filters (30 µm)
NAME
Sample Filter
TYPE
Miltenyi Biotec
BRAND
130-041-407
SKU

Count nuclei with DAPI or AO/PI depending on available counter. Make notes about sample quality if a lot of debris is still present then sorting will be needed later before starting single-cell.
FIXATION AND PERMEABILIZATION

Fix using 10x Genomics GEM-X Flex Sample Preparation v2 Kit (PN-1000781) based on 10x Genomics demonstrated protocol (CG000782 rev. D).
Centrifuge 500 x g, 4°C, 00:05:00 .
5m
Prepare Fixation Buffer B, maintain at room temperature.
  1. Conc. Fix & Perm Buffer B (10x Genomics PN-2001301) 1X.
  2. Formaldehyde 4%.
  3. Top off with Nuclease-free Water to 500 µL.
Remove the supernatant. Up to 30 µL might be left if working with >= 50,000 nuclei.
Resuspend nuclei in 500 µL room temperature Fixation Buffer B. Pipette mix 5x.
Incubate for one hour at Room temperature or 16-24h at 4 °C .
Note
Overnight fixation is recommended if samples will be stored long term (Up to 12 months).

Add 500 µL room temperature Additive C (10x Genomics PN-PN2001332) to the sample and pipette mix 5x.
Centrifuge 850 x g, Room temperature, 00:05:00 .
5m
Prepare Quenching Buffer B, maintain at 4 °C.
  1. Conc. Quench Buffer B (10x Genomics PN-2001300) 1X.
  2. Top off with Nuclease-free Water to 1000 µL.
Remove the supernatant. Up to 30 µL might be left if working with >= 50,000 nuclei.
Add 1 mL chilled Quenching Buffer B to the sample and pipette mix 5x, keep on On ice .
Proceed to the next step of nuclei sorting via FACS.

Important. Samples should be prepared and stored for long-term and processed later after one freeze-thaw cycle. It is recommended to collect all samples up to this point and sort them at once and proceed to Flex.
Long-term Storage at -80°C
  • Add 0.1 volume pre-warmed Enhancer (10x Genomics PN-2000482).
  • Add 50% glycerol for final concentration of 10%.
  • Store at -80 °C for up to 12 months.
Nuclei enrichment with FACS
Nuclei should be thawed according to 10x Genomics demonstrated protocol (CG000782 rev. D).
Resuspend nuclei in 500 µL Sorting & Collection buffer. Set aside around 5-10% of sample for negative control.
Nuclei can be stained either with DAPI or SYTO9.
  • To stain with DAPI, use 2.5 µL 200x DAPI stock into 500 µL sample.
  • To stain with SYTO9:
  1. Prepare intermediate SYTO9 solution by mixing 1 µL of 5 mM stock solution with 1 mL of PBS.
  2. Prepare working solution by mixing 100 µL of the intermediate stock to 900 µL of PBS.
  3. Centrifuge sample 500 x g, 4°C, 00:05:00 .
  4. Remove supernatant and resuspend in 1 mL SYTO9 working solution.
  5. Incubate 00:20:00 at room temperature.
  6. Wash once with 500 µL Sorting & Collection buffer.

Filter cells/nuclei through a 35 µm filter into Falcon Test Tube before sorting.
Equipment
Falcon™ Test Tube with Cell Strainer Snap Cap
NAME
Corning
BRAND
cat# 352235
SKU
https://www.fishersci.com/shop/products/falcon-tube-cell-strainer-cap-mesh-size-35um/0877123
LINK

After sorting immediately proceed to next steps. Next stopping point is only overnight Probe Hybridization during step 1 "Probe Hybridization" (see one of 10x Genomics GEM-X Flex Gene Expression User Guides).
Decrosslinking
1h
While sorting prepare buffers (Quantities for 1 sample):
  • 1.5 mL 10x Genomics Quenching Buffer B (Step 2.8) or 1.5 mL S&C Buffer.
  • 1 mL PBS-T.
  • Thaw aliquots 500 µL 162 nM 2-amino-5-methylbenzoic acid in PBS.
  • 20 µL 40 µg/mL Proteinase K in PBS (1:500 Stock dilution).
After sorting wash sample once:
Centrifuge 850 x g, 4°C, 00:05:00 , remove supernatant, re-suspend in 500 µL S&C Buffer.
5m
Prepare 500 µL decrosslinking buffer:
  • 463 µL 162 nM 2-amino-5-methylbenzoic acid in PBS for final conc. of 150 nM.
  • 31.25 µL 8M Urea for final conc. of 0.5 M.
  • 6.25 µL 40 µg/mL Proteinase K for final conc. of 0.5 µg/mL.
Centrifuge sample 850 x g, 4°C, 00:05:00 . Remove supernatant.
5m
Resuspend sample in 500 µL decrosslinking buffer. Pipette mix 5x.
Incubate sample on thermoblock (1.5mL) with heated lid:
  1. 80 °C 30 min.
  2. 22 °C 10 min.
40m
Alternatively, PCR tubes and thermal cycler could be used to same effect.
After decrosslinking wash sample twice:
Centrifuge 850 x g, 4°C, 00:05:00 , remove supernatant, re-suspend in 500 µL PBS-T.

5m
After washes re-suspend in Quenching Buffer.
Centrifuge 850 x g, 4°C, 00:05:00 , remove supernatant, re-suspend in 500 µL Quenching Buffer B.
5m
After decrosslinking immediately proceed to 10x Genomics Flex assay. Next stopping point is only overnight Probe Hybridization during step 1 "Probe Hybridization" (see one of 10x Genomics GEM-X Flex Gene Expression User Guides).
Protocol references
1. Ignas Masilionis, Ojasvi Chaudhary, Ronan Chaligne, Linas Mazutis 2022. Nuclei extraction for single-cell RNAseq from frozen tissue using Singulator 100. protocols.io https://protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-b7vxrn7n
2. Fixation of Cells & Nuclei for GEM-X Flex Gene Expression, 10x Genomics
3. GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples, 10x Genomics
4. GIFT-seq: Gapfill and Library Preparation Protocol