Nov 20, 2023
Nuclei extraction for 10x Genomics Single Cell Multiome ATAC + Gene Expression from frozen tissue using Singulator™ 100 or 200 (S2 Genomics) V2.0
Forked from a private protocol
- 1Single-Cell Analytics Innovation Lab, Memorial Sloan Kettering Cancer Center;
- 2Memorial Sloan Kettering Cancer Center
Protocol Citation: Ignas Masilionis, Ojasvi Chaudhary, Brigita Meskauskaite Urben, Ronan Chaligne 2023. Nuclei extraction for 10x Genomics Single Cell Multiome ATAC + Gene Expression from frozen tissue using Singulator™ 100 or 200 (S2 Genomics) V2.0. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkmq6l5r/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: May 06, 2022
Last Modified: November 20, 2023
Protocol Integer ID: 62125
Keywords: Single Cell, 10x Genomics, Nuclei, Multiome, Singulator, S2 Genomics, snRNAseq, snATACseq, Single Nuclei
Abstract
This protocol describes nuclei extraction from snap frozen tissue using Singulator, including washing nuclei suspension in buffer supplemented with sucrose, and nuclei FACS sorting. The resulting nuclei suspension is suitable for Single Cell Multiome ATAC + Gene Expression using 10x Chromium platform. The protocol has been validated on various healthy and cancer snap frozen human and mouse tissues (lung, brain, prostate, core needle biopsies etc) .
Guidelines
Make sure to use Swinging bucket centrifuges to spin nuclei.
Materials
MATERIALS
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306
Trizma hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2194-100ML
Sodium Chloride Solution 5 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #59222C
Magnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Digitonin (5%)Thermo FisherCatalog #BN2006
Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-FreeMerck MilliporeSigma (Sigma-Aldrich)Catalog #126615-25ML
7-AAD (7-Aminoactinomycin D)Thermo FisherCatalog #A1310
Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Protocol materials
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Materials
Magnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Materials
7-AAD (7-Aminoactinomycin D)Thermo FisherCatalog #A1310
Materials, Step 12
Digitonin (5%)Thermo FisherCatalog #BN2006
Materials
Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Materials, Step 3
Trizma hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2194-100ML
Materials
Sodium Chloride Solution 5 M Merck MilliporeSigma (Sigma-Aldrich)Catalog #59222C
Materials
Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-FreeMerck MilliporeSigma (Sigma-Aldrich)Catalog #126615-25ML
Materials
4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306
Materials
Preparation
Preparation
15m
Note
IMPORTANT
AFTER NUCLEI ISOLATION, YOU STILL NEED TO PERMEABILIZE THEM (step. 14) BEFORE TRANSPOSITION
- Turn on and pre-cool Singulator 00:10:00
- Make sure there is sufficient number of Nuclei cartridges (or NIC+ cartridges for samples <20mg) stored / pre-cooled at 4 °C
10m
Take the tissue out of LN2 or -80C freezer and place it on dry ice to keep it frozen
Ideally, piece should be <100mg (if not cut it in smaller pieces: <100mg and >10mg).
Cutting can be done with razor blade and within a petri dish on dry ice. Hold the tissue with cooled and RNAse free tweezers (be careful it might fly off). Work fast to not let the tissue thaw while cutting. Place tissue back in a tube on dry ice immediately after cutting.
10m
Prepare 2mL of Nuclei Wash Buffer per sample:
Tris-HCl (pH 7.4): 10 millimolar (mM) final
NaCl: 10 millimolar (mM) final
MgCl2: 3 millimolar (mM) final
Purified BSA: 1% final
DTT: 1 millimolar (mM) final
0.2 - 1.0 U/ul RNAse inhibitor (depending on tissue type, see RNase Activity in Mouse Tissue below) (Protector RNase InhibitorSigma AldrichCatalog #3335402001 )
Top-off to 2mL with Ambion Nuclease-free water
5m
Singulator
Singulator
10m
Add the following directly into Nuclei Isolation Cartridge before adding your tissue:
RNAse inhibitor (0.2-1.0 U/uL final depending on tissue type - Final volume ~ 3.5 mL for standard nuclei isolation protocol)
DTT, 1mM final concentration (3.5uL of 1 Molarity (M) DTT for standard nuclei isolation protocol).
Transfer tissue into the cooled Nuclei Isolation Cartridge and start standard nuclei isolation protocol.
10m
Washes and Prep for FACS
Washes and Prep for FACS
15m
When run is over on Singulator, transfer nuclei suspension (~3.5mL for standard protocol) into 5mL Protein LoBind eppendorf Tubes (or split 3.5mL evenly into four 1.5mL Protein LoBind eppendorf Tubes, conical bottom of the 1.5mL and 5mL tubes increase nuclei recovery).
5m
Add 500uL 2 Molarity (M) Sucrose solution to a tube (250 millimolar (mM) final conc). Adjust volume if using 1.5mL tubes.
Invert the tube slowly at least 10 times to mix sucrose and nuclei suspension (Do not vortex!).
Make sure sucrose is well mixed, if sucrose cushion is visible after centrifugation, mix again and repeat centrifugation.
500 x g, 4°C, 00:05:00 , Swinging bucket centrifuge
5m
Nuclei Washes and FACS
Nuclei Washes and FACS
1h
After centrifugation, a layer of debris might be visible at the top and a pellet of nuclei at the bottom (for lower yields it might not be noticeable).
Aspirate supernatant with a P1000 pipette, be really careful when removing the debris layer on the meniscus without disrupting nuclei pellet at the bottom of the tube (some liquid can be left to prevent pellet aspiration ~50uL).
2m
Re-suspend Nuclei pellet in ~400uL Nuclei Wash Buffer.
This can be adjusted based on tissue input and pellet size.
1m
OPTIONAL:
A second wash with Nuclei Wash buffer can be performed to remove traces of sucrose (as it might impact FACS sorting).
A second wash with 250mM sucrose can be performed if high amount of debris or fat is still present in the sample.
Filter nuclei suspension through a 35 μm FACS tube cap filter (blue) or 30 μm Pre-separation filters (Miltenyi).
2m
Count nuclei using DAPI on automated cell counter (Countess II for instance)
Trypan can also be used to count in Bright field, but in our experience DAPI count is more accurate.
5m
Nuclei should be stained with 7-AAD (7-Aminoactinomycin D)Sigma AldrichCatalog #A1310 , for sorting. Positive nuclei population can be FACS into 500uL of Nuclei wash buffer using 1.5mL Lobind Eppendorf as collection tube (see attached PDF for gating setup example).
Suggested nozzle size: 100um.
Ideally, aim to collect >200 000 nuclei.
Sort_Report_DAPI_pos_Nuclei.pdf 
30m
10x Genomics
10x Genomics
35m
Concentrate the nuclei suspension post-sorting:
500 x g, 4°C, 00:05:00 , Swinging bucket centrifuge
Then, aiming for >=1000 nuclei/uL using FACS numbers, re-suspend sorted nuclei with nuclei Wash buffer .
Count nuclei using DAPI on Countess II cell counter.
Trypan can also be used to count in Bright field, but DAPI counts are more accurate.
5m
Aliquot 200k nuclei in 0.2mL PCR tube.
500 x g, 4°C, 00:05:00 , Swinging bucket centrifuge
Remove supernatant leaving 5uL precisely (calculate: (Aliquot volume minus 5uL) and aspirate that amount, eyeballing it is highly discouraged).
Then continue to 10X Genomics Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing protocol CG000365. Following "Low Cell Input Nuclei Isolation" in Appendix starting at step e., omit NP40 from lysis buffer and perform lysis for 30s.
30m