Dec 12, 2024

Public workspaceNuclei isolation from human frozen adipose tissue for single-nuclei RNA-sequence. Adapted from three published articles. V.2

DOI
dx.doi.org/10.17504/protocols.io.eq2ly6w5qgx9/v2
  • 1University of Minnesota
  • Cellular Senescence Network (SenNet) Method Development Community
Mythili Dileepan
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly6w5qgx9/v2
Protocol Citation: Mythili Dileepan, David Bernlohr, Paul Robbins, Laura Niedernhofer 2024. Nuclei isolation from human frozen adipose tissue for single-nuclei RNA-sequence. Adapted from three published articles.. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6w5qgx9/v2Version created by Mythili Dileepan
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, 2024
Last Modified: December 12, 2024
Protocol Integer ID: 115073
Funders Acknowledgements:
NIH Common Fund
Grant ID: U54 AG076041
Abstract
This is a protocol for isolating nuclei from human adipose tissue and it was adapted from three published articles.
Nuclei isolation from human frozen adipose tissue for single-nuclei RNA-sequence
Nuclei isolation from human frozen adipose tissue for single-nuclei RNA-sequence
First essential step
  1. Freshly prepare all reagents on the day of isolation.
  2. Wipe all surfaces and equipment with RNAse Zap
  3. Make sure the centrifuge is at 4° C
Buffer Preparation 2x ST (It can be prepared previous evening and kept at room temperature)
ABCD
Reagent Stock Concentration 2X ST Buffer Concentration Volume for 50 mL of 2X ST Buffer
NaCl 5 M 292 mM 2.92 mL
Tris 1 M 20 mM 1 mL
CaCl2 1 M 2 mM 100 μL
MgCl2 1 M 42 mM 2.1 mL
H2O - - 43.88 mL
TST (2 mL should be prepared for each tissue sample) (homogenization buffer) *10% Tween-20 can be prepared ahead of time and stored at 4ºC
AB
Reagent Volume for 2 mL Working Solution (per Sample)
2x ST stock solution 1000 μL
BSA Stock Solution (10%) 2 μL (0.01% final concentration)
10% Tween-20* 6 μL
H2O 636 μL
Superase RNase Inhibitor (20U/μL) 20 μL (1 U/μL final concentration)
Sucrose (1.5M) 334 ul
Protease inhibitor (100x) 2 ul



1x ST
AB
Reagent Volume for 3.5 mL Working Solution (per Sample)
2x ST stock solution 1748 μL
H2O 1717 μL
Ribolock RNase Inhibitor (40 U/μL) 35 μL
PBS + BSA (1%) + RNase Inhibitors
AB
Reagent  (per Sample)
PBS 1X 3500 μL
BSA Stock Solution (10%) 400 μL
Protector RNase Inhibitor (40 U/μL) 100 μL
Tissue Dissociation
  • Fill a GentleMACS C tube with 2 mL of TST buffer per sample. Keep tubes on wet ice.
  • Transfer a ~130-150mg piece of adipose tissue directly into the buffer.
  • Make sure that tissues are floating freely in buffer and not sticking onto the walls of the tube.
  • Secure the C tubes to the GentleMACS Dissociator and run the “mr_adipose tissue, 35 sec” program.
  • Repeat the program for three runs on each tissue sample.
  • Detach the tubes from the GentleMACS Dissociator and Incubate the samples on wet ice for 10 minutes (upside down).
  • Set the acceleration to 10 and deceleration to 5
  • Centrifuge the tubes for 2 minutes at 500g at 4ºC to collect the suspension and remove any foam.
  • Immediately after centrifugation, resuspend the nuclei pellet in the supernatant within the same C tube. Pipette up and down 30 times.

3. Filtration
  • Place a 50 mL Falcon tube on wet ice with a 40 um Falcon Cell Strainer.
  • First wash the filter with 1 mL of 1x ST buffer with RNase inhibitors.
  • Transfer the dissociated ~2 mL suspension of nuclei into the filter.
  • Wash the used C tube with 1 mL of 1x ST buffer with RNase inhibitors, then transfer it also into the filter.
  • Lastly, wash the filter with an additional 1 mL of 1x ST with RNase inhibitors.
4. Centrifugation and nuclei isolation
  • Centrifuge in a swinging bucket rotor for 10 minutes at 2500 g and 4ºC.
  • Carefully remove the supernatant by pipetting, leaving approximately 20 µL behind in the tube.
  • Wash the nuclear pellet 2-3 times with 1000 µL of PBS + BSA + RNase Inhibitor solution. Gently pipette up and down 30 times before each centrifugation.
  • Resuspend the remaining nuclear pellet in 100 µL of PBS + 1% BSA + RNase Inhibitor solution.
  • Submit the nuclei sample immediately for QC and cDNA preparation.
Adapted from three published articles.
Adapted from three published articles.
1. Emont, M.P., Jacobs, C., Essene, A.L. et al. A single-cell atlas of human and mouse white adipose tissue. Nature 603, 926–933 (2022). https://doi.org/10.1038/s41586-022-04518-2

2. Whytock KL, Divoux A, Sun Y, Hopf M, Yeo RX, Pino MF, Yu G, Smith SR, Walsh MJ, Sparks LM. Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling. STAR Protoc. 2023 Mar 17;4(1):102054. doi: 10.1016/j.xpro.2023.102054. Epub 2023 Jan 20. PMID: 36853719; PMCID:PMC9876942.

3. TST Nuclei Isolation with GentleMACS – 220301 Sébastien Vigneau1
DOI dx.doi.org/10.17504/protocols.io.dm6gp39z1vzp/v1
1Dana-Farber Cancer Institute