Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics --University of Minnesota TMCs
Laura J Niedernhofer, David A Bernlohr, Ann Hertzel
Abstract
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
Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics.pdf
Before start
General preparation
Timing: 1 h
- Clean Dounce homogenizers and pre-cool them on ice CRITICAL CAL: If nuclei are to be used for transcriptomics, rinse Dounce homogenizers with
RNaseZAP and DEPC-treated water to minimize RNase activity.
-
Pre-cool centrifuges to 4C.
-
Pre-cool tubes for nuclei isolation and Petri dishes for tissue mincing on Note:
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.
- 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
| A | B | C |
|---|---|---|
| Chemicals, peptides and recombinant proteins | ||
| 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) | Lonza | Cat#BE17-737E; CAS: 7365-45-9 |
| Bovine Serum Albumin | Sigma-Aldrich | Cat#B6917; CAS: 9048-46-8 |
| Diethyl dicarbonate (DEPC) | Sigma-Aldrich | Cat#D5758; CAS: 1609-47-8 |
| DL-Dithiothreitol (DTT) | New England Biolabs | Cat#B1034A; CAS: 3483-12-3 |
| IGEPAL CA-630 | Sigma-Aldrich | Cat#I8896; CAS: 9002-93-1 |
| Magnesium chloride (MgCl2) | Sigma-Aldrich | Cat#M1028; CAS: 7786-30-3 |
| Phosphate buffered saline (PBS) (10×, pH 7.2) | Gibco | Cat#70013-016 |
| Potassium chloride (KCl) | Sigma-Aldrich | Cat#P9541; CAS: 7447-40-7 |
| RNase Inhibitor, Murine | New England Biolabs | Cat#M0314 |
| RNaseZAP | Sigma-Aldrich | Cat#R2020 |
| Sucrose | Sigma-Aldrich | Cat#S0389; CAS: 57-50-1 |
| Trypan Blue | Bio-Rad | Cat#1450013 |
| Biological Samples | ||
| Mouse white adipose tissues | N/A | N/A |
| Critical commercial assays | ||
| Chromium Next GEM Chip G Single Cell Kit (48 rxns) | 10x Genomics | Cat#1000120 |
| Chromium Next GEM Single Cell 3’ Kit v3.1 (16 rxns) | 10x Genomics | Cat#1000268 |
| Dual Index Kit TT Set A (for Gene Expression Libraries) | 10x Genomics | Cat#1000215 |
| Experimental models: organisms/strains | ||
| Mus musculus C57BL/6J and C57BL/6N | N/A | N/A |
| Other | ||
| 0.2 μm Syringe filters | Sartorius | Cat#17845-ACK |
| 1.5 mL DNA LoBind tubes | Eppendorf | Cat#0030108051 |
| 7 mL Dounce homogenizer | Sigma-Aldrich | Cat#D9063 |
| 5 mL DNA LoBind tubes | Eppendorf | Cat#0030122348 |
| 6 cm Petri dish | Thermo Scientific | Cat#150288 |
| 50 mL Tube | SARSTEDT | Cat#62.547.254 |
| 10× Genomics Chromium Controller | 10x Genomics | Cat#PN110203; RRID:SCR_019326 |
| BRAND counting chamber BLAUBRAND Bürker pattern | Sigma-Aldrich | Cat#BR718920 |
| Flowmi Cell Strainer, porosity 40 μm | Sigma-Aldrich | Cat#BAH136800040 |
| NovaSeq 6000 System | Illumina | Cat#20012850; RRID:SCR_016387 |
| pluriStrainer 70 μm (Cell Strainer) | pluriSelect | Cat#43-50070-51 |
| Surgical Scalpel Blade No.22 | Swann-Morton | Cat#0208 |
Materials and equipment
Nuclei isolation buffer (NIB)
| A | B | C |
|---|---|---|
| Sucrose (0.5 M) | 250 mM | 500 μL |
| HEPES (1 M) | 10 mM | 10 μL |
| MgCl2 (150 mM) | 1.5 mM | 10 μL |
| KCl (2 M) | 10 mM | 5 μL |
| IGEPAL CA-630 (1%) | 0.001% | 1 μL |
| DTT (0.1 M) | 0.2 mM | 2 μL |
| RNase inhibitor (40,000 U/mL) | 0.5 U/μL | 12.5 μL |
| DEPC-treated water | N/A | 459.5 μL |
| Total | N/A | 1 mL |
Nuclei resuspension buffer (NRB)
| A | B | C |
|---|---|---|
| BSA in 1× PBS (5%) | 1% | 200 μL |
| MgCl2 (150 mM) | 2 mM | 13.3 μL |
| RNase inhibitor (40,000 U/mL) | 0.04 U/μL | 1 μL |
| PBS (1×) | N/A | 785.7 μL |
| Total | N/A | 1 mL |
Steps
Tissue Homogenization
Transfer 400 mg of gonadal adipose tissue (or 100 mg of inguinal adipose tissue) to a pre-cooled Petri dish on ice (Figure 1A).
Add 500 µL of NIB and mince the tissue thoroughly (<1 mm3 ) using a scalpel (Figures 1B and 1C).
With the scalpel, transfer the minced adipose tissue to a pre-cooled 7 mL glass Dounce homogenizer on ice (Figure 1D).
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).
Pre-wet a 70 µm cell strainer with DEPC-treated water.
Filter the homogenate through the pre-wetted cell strainer into a pre-cooled 50 mL tube on ice.
Wash the Dounce homogenizer with 1 mL NIB and filter through the same cell strainer.
Transfer the filtered homogenate to a pre-cooled 5 mL DNA Low binding tube on ice using a P1000 pipette.
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).
Isolation of Nuclei
Centrifuge the homogenate at 1000 x g for 10 min at 4°C using a fixed-angled rotor (Figure 2A).
Aspirate the lipid layer using a vacuum pump (Figure 2B).
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.
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).
Pellet the nuclei by centrifuging at 500 x g for 10 min at 4°C using a swing-bucket rotor (Figure 2D).
Aspirate the supernatant.
Use vacuum aspiration to remove most of the supernatant (leave 80–100 mL supernatant) and remove remaining supernatant with a P100 pipette.
Wash the pellet - Resuspend the nuclei pellet in 750 µL of NIB
Pellet the nuclei by centrifuging at 500 x g for 10 min at 4°C
Resuspend the nuclei pellet in 100 µL NRB by pipetting using a regular P1000 pipette.
Pre-wet a 40 mm tip strainer with NRB.
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.
Proceed to nuclei counting using a Bürker counting chamber and Trypan Blue.
Mix nuclei and Trypan Blue in equal ratio in a separate tube.
Transfer 10 µL of the nuclei-Trypan Blue solution to the Bürker counting chamber.
Count at least 3 squares using bright-field microscopy and quantify the nuclei concentration.
