Oct 01, 2020
Nuclei Isolation from Human Frozen Liver
- 1[Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States];
- 2[Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States;
- 3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States]
Protocol Citation: Sanjay Subramanian, Stacey S Huppert 2020. Nuclei Isolation from Human Frozen Liver. protocols.io https://dx.doi.org/10.17504/protocols.io.bhpej5je
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: June 18, 2020
Last Modified: October 01, 2020
Protocol Integer ID: 38342
Abstract
This protocol is used to isolate human liver nuclei from frozen samples in an effort to facilitate human nuclei extraction for use in single nuclear RNA sequencing and single cell ATAC sequencing without the need for fresh samples. This approach enables the ability to acquire signatures from all cell types, even those from fibrotic liver samples. A brief fixation step is included in order to preserve nuclear membrane integrity and prevent nuclear leakage. This protocol has been tested and works in frozen human liver samples weighing approximately 1 gram stored in cryopreservation medium. Single nuclear RNA seqencing was run using the 10x Chromium system.
This project was supported in part by NIH P30 DK078392 (Gene Analysis Core) of the Digestive Diseases Research Core Center in Cincinnati, R01DK107553, and the Cincinnati Pediatric Cell Atlas Center funded by Cincinnati Children’s Research Foundation to Stacey S. Huppert
Guidelines
Sample Storage
Samples are prepared by incubating fresh tissue chunks in HypoThermosol® FRS solution for 15 minutes on ice, followed by a 30 minute incubation in CryoStor® CS10 cryopreservation medium on ice. Samples are then frozen slowly in a cryo-freezing container overnight at -80 °C and subsequently transferred to liquid nitrogen storage.
10x PBS (1L)
80g NaCl
2g KCl
11.5g Na2HPO4 7H2O
2g KH2PO4
1L diWater
1x PBS with 1% BSA
Make up 100mL 1x PBS by diluting 10mL 10x PBS in 90mL MilliQ water
Make up 100mL 1% BSA in 1x PBS by dissolving 1g BSA in 100mL 1x PBS
4% PFA
10mL of 16% Paraformaldehyde
30mL of 1x PBS
2x Stock ST Buffer (20mL)
| Reagent | Volume | Final Concentration | |
| 5M NaCl | 1.168mL | 292mM | |
| 1M Tris-HCl pH 7.5 | 400μL | 20mM | |
| 1M CaCl2 | 40μL | 2mM | |
| 1M MgCl2 | 840μL | 42mM | |
| 1x PBS with 1% BSA | 17.548mL | ||
| SUPERaseIN | 4μL |
1x ST Buffer (5mL)
2.5mL of 2x Stock ST Buffer
2.5mL of 1x PBS with 1% BSA
1x ST Buffer with 0.1% PFA (5mL)
2.5mL of 2x Stock ST Buffer
125μL of 4% PFA
2.374mL of 1x PBS with 1% BSA
1uL of SUPERaseIN
TST Homogenization Solution (5mL)
2.5mL of 2x Stock ST Buffer
120μL of Tween-20
2.375mL of 1x PBS with 1% BSA
5μL of RNase inhibitor
Materials
MATERIALS
Paraformaldehyde, 16% (wt/vol)Electron Microscopy SciencesCatalog #15710
CryoStor® CS10 100 mL
Stemcell TechnologiesCatalog #7930
Magnesium chloride solution for molecular biology (1.00 M)Sigma – AldrichCatalog #M1028
Recombinant RNAse InhibitorTakarabioCatalog #2313A
TWEEN® 20Sigma AldrichCatalog #P7949
NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
HypoThermosol® FRS Preservation solution Sigma AldrichCatalog #H4416
Falcon™ Cell Strainers, Mesh size: 70um; whiteThermo FisherCatalog #087712
UltraPure™ 1 M Tris-HCI Buffer, pH 7.5Thermo FisherCatalog #15567027
SUPERase• In™ RNase Inhibitor (20 U/μL)Thermo FisherCatalog #AM2696
40um Cell Strainer Fisher ScientificCatalog #22363547
Albumin Bovine Fraction V (BSA)Research Products International (rpi)Catalog #A30075
1 mL Tissue Grinder DounceDuran Wheaton KimbleCatalog #357538
Calcium chloride 1 M in aqueous solutionVwrCatalog #97062-820
Thaw small tissue sample from frozen preservation medium at room temperature and wash in 1x PBS
Briefly mince tissue into small chunks using a scalpel and/or razor in petri dish on ice
Homogenize chunks using Tissue Grinder Dounce with Pestle A in 500μL to 1mL of ice-cold TST buffer
1 mL TST buffer
Note
1mL Tissue Grinder Dounce comes with two pestles with varying clearance. Pestle "A" is best suited for this protocol
Note
Ensure thorough homogenization, even after solution seems sufficiently aqueous. Quality of fixation depends on achieving full dissociation of cellular membranes
Filter suspension through a 70μM cell strainer and bring total volume up to 5mL with ice-cold ST buffer with 0.1% PFA. Ensure solution is mixed well to circulate fix
5 mL ST buffer with 0.1% PFA
Centrifuge nuclei at 500g for 5min at 4 °C
00:05:00 centrifuge 500g at 4 °C
Remove supernatant and resuspend in 5 mL ST buffer
5 mL ST buffer
Repeat steps 5-6
Note
Final resuspension volume may be greater or less than stated and should be judged accordingly based on pellet size
Filter solution through a 40μM cell strainer
Analyze nuclei morphology using hemocytometer and Trypan blue
Adjust nuclei concentration as needed (700-1200 cells/μL for 10x Chromium system)
1000 nuclei/μL