Nov 06, 2025

Single nuclear isolation

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw421wlmk/v1
  • Daniel Alligood1,
  • Alex Park1,
  • Jun Guo1
  • 1Department of Surgery, Washington University in St. Louis
Daniel Alligood
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw421wlmk/v1
Protocol Citation: Daniel Alligood, Alex Park, Jun Guo 2025. Single nuclear isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw421wlmk/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: October 09, 2025
Last Modified: November 06, 2025
Protocol  Integer ID: 229438
Keywords: single nuclear isolation single nuclear isolation protocol, single nuclear isolation, single nuclear isolation protocol, mouse liver tissue, rnalater, liquid nitrogen
Abstract
Single nuclear isolation protocol adapted from Nadelmann, et. al. doi: 10.1002/cpz1.132. This protocol has been tested on mouse liver tissue stored in RNALater and flash frozen in liquid nitrogen then stored in -80C freezer.
Reagent Preparation
Nuclei isolation buffer 2 (NIM2)
For 1 sample,

  • NIM1 (see above) - 1246.25 ul
  • 1 mM DTT (Fisher Scientific PI20290) - 1.25 ul - 1uM
  • 50x Protease Inhibitor (Sigma 11873580001) - 25 ul

total volume: 1250 ul
Nuclei isolation buffer 1 (NIM1)
For 1 sample,

  • 1.5 M sucrose (Sigma @0389) - 625 ul - 250mM
  • 2 M KCl (Ambion AM9640G) - 46.875 ul - 25mM
  • 1 M MgCl2 (Ambion AM9530G) - 18.75 ul - 5 mM
  • 1 M TrisCl, pH 8 (Ambion AM9855G) - 37.5 ul - 10 mM
  • Nuclease-free water - 3021.88 ul

total volume: 3750 ul

Homogenization buffer (HB)
For 1 sample,

  • NIM2 (see above) - 1212.5 ul
  • 40 U/ul RNaseIn (Ambion AM2682) - 12.5 ul - 0.4 U/ul
  • 20 U/ul SuperaseIn (Ambion AM2694) - 12.5 ul - 0.2 U/ul
  • 10% (v/v) Triton X-100 (Sigma T8787) - 12.5 ul - 0.10%

total volume: 1250 ul
Storage buffer (SB)
For 1 sample,

  • Phosphate-buffered saline (Leinco Technologies P349) - 995ul
  • Bovine serum albumin (Sigma A9647) - 40mg - 4%
  • 40 U/ul Protector RNaseIn (Sigma 03 335 402 001)- 5 ul - 0.2 U/ul

total volume: 1000ul
Sample Preparation
At harvest, store samples in RNALater (Invitrogen AM7021) and flash freeze in liquid nitrogen. Store in -80C freezer.
Nuclei Isolation
10m
Thaw RNALater stored tissue on ice.
Nuclei Isolation
10m
Remove tissue with transfer pipette and place into a wide-based 2ml Eppendorf tube with 1000ml of homogenization buffer. (use sharp dissection to break tissue into smaller chunks)
Place 2 large (3mm) and 3 small (2.3mm) homogenization beads
Set temperature of tissue homogenizer (Servicebio SWE-3D) to -20C. Homogenize tissue at 35Hz for 60s. (Difficult tissue may be run up to 60Hz for 60s; do not run more than twice).
All samples to lyse on ice for 5 minutes
5m
Filter homogenized tissue through 40-um cell strainer into a non-stick RNAase-free 1.5ml tube
Centrifuge 500g for 5 minutes at 4C
5m
transfer the supernatant into new 1.5ml RNAase-free tubes (can be used for bulk RNA sequencing)
Resuspend nuclei pellet with 500ul of SB
Filter 250ul into FACS tubes
Spin FACS tube down at 200g for 20s at 4C
Choose a negative control (typically a sham); separate 100ul of sample into a new FACS tube
Add 2 drops of nucblue in all FACS tubes but control
Vortex 5s and let the samples sit for 15 minutes
Flow Cytometry
Run the negative control to set up the gate
Gating strategy: SSC-A vs FSC-A --> SSC-H vs SSC-W --> FSC-H vs FSC-W --> Hoechst-A vs SSC-A (doublet strands are typically seen)
Sort the entire sample to maximize yield (approximately 30 minutes) into 1.5ml non-stick RNase-free micro centrifuge tubes. Keep on ice until all sorting is completed.
Counting Nuclei
5m
Centrifuge samples 500g for 5min at 4C
5m
Remove supernatant carefully (save supernatant)
Re-suspend nuclei pellet according to FACS Cell numbers
  • <50,000 - 30 ul
  • 50,000-100,000 - 50ul
  • 100,000-150,000 - 70 ul
  • 150,000+ - 90ul
Count nuclei using Countess. Add 6ul of sample with 6ul of trypan blue then load 10ul of solution into Countess chamber. OPTIONAL use automatic cell concentration device. (ideal concentration is 8x105 - 1.2x106)
Protocol references
Nadelmann, E. R.,  Gorham, J. M.,  Reichart, D.,  Delaughter, D. M.,  Wakimoto, H.,  Lindberg, E. L.,  Litviňukova, M.,  Maatz, H.,  Curran, J. J.,  Ischiu Gutierrez, D.,  Hübner, N.,  Seidman, C. E., &  Seidman, J. G. (2021).  Isolation of nuclei from mammalian cells and tissues for single-nucleus molecular profiling. Current Protocols,  1, e132. doi: 10.1002/cpz1.132