TITLE: Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics --University of Minnesota TMCs
AUTHORS: Ann Hertzel, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
This protocol describes the steps to isolate and obtain total nuclei from frozen mouse white adipose tissues (WAT). We utilized this protocol to obtain nuclei from 4- and 24- month-old mouse epididymal adipose samples. Additionally this protocol can be used for various other murine adipose depots, with adjustment of the final resuspension volume to accommodate variations in the size of the final nuclei pellet as a result of the nuclei yield.

Adapted from STAR Protocols - Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics
  


[BEFORE_START]
General preparation
Timing: 1 h
1. Clean Dounce homogenizers and pre-cool them on ice.
CRITICAL: If nuclei are to be used for transcriptomics, rinse Dounce homogenizers with
RNaseZAP and DEPC-treated water to minimize RNase activity.
2. Pre-cool centrifuges to 4C.
3. Pre-cool tubes for nuclei isolation and Petri dishes for tissue mincing on ice.
Note: For each isolation, prepare 13 50 mL tube, 23 5 mL DNA low binding tubes, 23 1.5 mL
DNA low binding tubes, and 13 Petri dish.
4. Prepare nuclei isolation buffer (NIB) and nuclei resuspension buffer (NRB).
a. Filter buffers using a 0.2 mm syringe filter and pre-cool buffers on ice.
Note: Buffers are prepared freshly in order to minimize RNase activity.

Key resources table
REAGENT or RESOURCESOURCEIDENTIFIERChemicals, peptides, and recombinant proteins4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)LonzaCat#BE17-737E; CAS: 7365-45-9Bovine Serum AlbuminSigma-AldrichCat#B6917; CAS: 9048-46-8Diethyl dicarbonate (DEPC)Sigma-AldrichCat#D5758; CAS: 1609-47-8DL-Dithiothreitol (DTT)New England BiolabsCat#B1034A; CAS: 3483-12-3IGEPAL CA-630Sigma-AldrichCat#I8896; CAS: 9002-93-1Magnesium chloride (MgCl2)Sigma-AldrichCat#M1028; CAS: 7786-30-3Phosphate buffered saline (PBS) (10×, pH 7.2)GibcoCat#70013-016Potassium chloride (KCl)Sigma-AldrichCat#P9541; CAS: 7447-40-7RNase Inhibitor, MurineNew England BiolabsCat#M0314RNaseZAPSigma-AldrichCat#R2020SucroseSigma-AldrichCat#S0389; CAS: 57-50-1Trypan BlueBio-RadCat#1450013Biological samplesMouse white adipose tissuesN/AN/ACritical commercial assaysChromium Next GEM Chip G Single Cell Kit (48 rxns)10x GenomicsCat#1000120Chromium Next GEM Single Cell 3’ Kit v3.1 (16 rxns)10x GenomicsCat#1000268Dual Index Kit TT Set A (for Gene Expression Libraries)10x GenomicsCat#1000215Experimental models: organisms/strainsMus musculus C57BL/6J and C57BL/6NN/AN/AOther0.2 μm Syringe filtersSartoriusCat#17845-ACK1.5 mL DNA LoBind tubesEppendorfCat#00301080512 mL Dounce homogenizerSigma-AldrichCat#D89385 mL DNA LoBind tubesEppendorfCat#00301223486 cm Petri dishThermo ScientificCat#15028850 mL TubeSARSTEDTCat#62.547.25410× Genomics Chromium Controller10x GenomicsCat#PN110203; RRID:SCR_019326BRAND counting chamber BLAUBRAND Bürker patternSigma-AldrichCat#BR718920Flowmi Cell Strainer, porosity 40 μmSigma-AldrichCat#BAH136800040NovaSeq 6000 SystemIlluminaCat#20012850; RRID:SCR_016387pluriStrainer 70 μm (Cell Strainer)pluriSelectCat#43-50070-51Surgical Scalpel Blade No.22Swann-MortonCat#0208


Materials and equipment

Nuclei isolation buffer (NIB)
ReagentFinal concentrationAmountSucrose (0.5 M)250 mM500 μLHEPES (1 M)10 mM10 μLMgCl2 (150 mM)1.5 mM10 μLKCl (2 M)10 mM5 μLIGEPAL CA-630 (1%)0.001%1 μLDTT (0.1 M)0.2 mM2 μLRNase inhibitor (40,000 U/mL)0.5 U/μL12.5 μLDEPC-treated waterN/A459.5 μLTotalN/A1 mL

Nuclei resuspension buffer (NRB)
ReagentFinal concentrationAmountBSA in 1× PBS (5%)1%200 μLMgCl2 (150 mM)2 mM13.3 μLRNase inhibitor (40,000 U/mL)0.04 U/μL1 μLPBS (1×)N/A785.7 μLTotalN/A1 mL
 
 
Note: NIB and NRB are prepared freshly, filtered, and kept on ice until use to minimize RNase activity. Sucrose is prepared freshly in DEPC-treated water. IGEPAL CA-630 solution is diluted in DEPC-treated water and can be stored for several months at 20°C–25°C. BSA is dissolved in 1× PBS (in DEPC-treated water) and can be stored at −20°C. 1× PBS solution is prepared by diluting a 10× PBS solution in DEPC-treated water.
Alternatives: In principle, all reagents and resources listed in the key resources table can be replaced with equivalent items from other suppliers; however, the impact of alternative reagents on protocol performance has not been tested.

[STEPS]
SECTION: Tissue Homogenization
1. 
 

Transfer 400 mg of gonadal adipose tissue (or 100 mg of inguinal adipose tissue) to a pre-cooled Petri dish on ice (Figure 1A).
SECTION: Tissue Homogenization
4. Homogenize the tissue to release nuclei by applying 5 strokes with pestle A (loose) followed by 5
strokes with pestle B (tight) (Figures 1E and 1F).
 

 

SECTION: Tissue Homogenization
2. Add 500 µL of NIB and mince the tissue thoroughly (<1 mm3 ) using a scalpel (Figures 1B and 1C). 
 

SECTION: Tissue Homogenization
3. With the scalpel, transfer the minced adipose tissue to a pre-cooled 7 mL glass Dounce homogenizer on ice (Figure 1D).
SECTION: Tissue Homogenization
5. Pre-wet a 70 µm cell strainer with DEPC-treated water.
SECTION: Tissue Homogenization
6. Filter the homogenate through the pre-wetted cell strainer into a pre-cooled 50 mL tube on ice.
SECTION: Tissue Homogenization
7. Wash the Dounce homogenizer with 1 mL NIB and filter through the same cell strainer.
SECTION: Tissue Homogenization
8. Transfer the filtered homogenate to a pre-cooled 5 mL DNA Low binding tube on ice using a P1000 pipette.
SECTION: Tissue Homogenization
9. Wash the cell strainer with 1 mL NIB and add to the filtered homogenate in the 5 mL tube to a total volume of ∼2.5 mL using a P1000 pipette (Figure 1G).
SECTION: Isolation of Nuclei
10. Centrifuge the homogenate at 1000 x g for 10 min at 4°C using a fixed-angled rotor (Figure 2A). 
 

SECTION: Isolation of Nuclei
11. Aspirate the lipid layer using a vacuum pump (Figure 2B). 
 

SECTION: Isolation of Nuclei
12. Resuspend the pellet in the remaining supernatant using a P1000 pipette and transfer the resuspended nuclei to a new pre-cooled 5 mL DNA Low binding tube on ice.
SECTION: Isolation of Nuclei
13. To make sure that all nuclei are transferred, wash the first 5 mL tube twice with 1 mL NIB and transfer any remaining nuclei to the new 5 mL tube containing the resuspended nuclei using a P1000 pipette (Figure 2C).
SECTION: Isolation of Nuclei
14. Pellet the nuclei by centrifuging at 500 x g for 10 min at 4°C using a swing-bucket rotor (Figure 2D).
SECTION: Isolation of Nuclei
15. Aspirate the supernatant. 
 

SECTION: Isolation of Nuclei
18. Resuspend the nuclei pellet in 100 µL NRB by pipetting using a regular P1000 pipette.

 

SECTION: Isolation of Nuclei
19. Pre-wet a 40 mm tip strainer with NRB.
SECTION: Isolation of Nuclei
15.1. Use vacuum aspiration to remove most of the supernatant (leave 80–100 mL supernatant) and remove remaining supernatant with a P100 pipette. 
SECTION: Isolation of Nuclei
16. Wash the pellet - Resuspend the nuclei pellet in 750 µL of NIB
SECTION: Isolation of Nuclei
17. Pellet the nuclei by centrifuging at 500 x g for 10 min at 4°C
SECTION: Isolation of Nuclei
20. Filter the nuclei through the pre-wetted tip strainer using a P1000 pipette into a pre-cooled 1.5 mL DNA Low binding tube on ice (Figure 2E) to remove any remaining debris and avoid nuclei aggregation. 
 

SECTION: Isolation of Nuclei
21. Proceed to nuclei counting using a Bürker counting chamber and Trypan Blue. 
SECTION: Isolation of Nuclei
21.1. Mix nuclei and Trypan Blue in equal ratio in a separate tube. 
SECTION: Isolation of Nuclei
21.2. Transfer 10 µL of the nuclei-Trypan Blue solution to the Bürker counting chamber. 
SECTION: Isolation of Nuclei
21.3. Count at least 3 squares using bright-field microscopy and quantify the nuclei concentration.