Protocol Citation: Ann Hertzel, Laura J Niedernhofer, David A Bernlohr 2023. Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics --University of Minnesota TMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx56xgx1/v1
Manuscript citation:
CITATION
Van Hauwaert EL, Gammelmark E, Sárvári AK, Larsen L, Nielsen R, Madsen JGS, Mandrup S (2021). Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics..
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 06, 2023
Last Modified: November 29, 2023
Protocol Integer ID: 88926
Funders Acknowledgement:
NIH
Grant ID: 5U54AG079754-02
NIH
Grant ID: 5U54AG076041-03
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.pdf2.6MB
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.
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)
Reagent
Final concentration
Amount
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)
Reagent
Final concentration
Amount
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
Note
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.
Note
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.
Tissue Homogenization
Tissue Homogenization
30m
Note
To minimize contamination with blood cells from the vasculature, it is recommended to
do cardiac perfusion with 13 PBS in DEPC-treated water (typically 12 mL per perfusion) prior
to isolating the tissue.
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).
Note
CRITICAL: Nuclei yield will be reduced if the tissue is not finely minced.
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).
Note
CRITICAL: Homogenize carefully to minimize generation of heat and foam, which will
impact nuclei intactness.
Note
Note: For adipose tissues from obese mice, use the same amount of tissue as for lean tissues, but homogenize in a total volume of 1 mL NIB and apply 10 strokes with pestle A followed by 5 strokes with pestle B.
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
Isolation of Nuclei
2h
Centrifuge the homogenate at 1000 x g for 10 min at 4°C using a fixed-angled rotor (Figure 2A).
Note
Nuclei are located as a smear along the side of the 5 mL tube.
Aspirate the lipid layer using a vacuum pump (Figure 2B).
Note
CRITICAL: Only remove the top lipid layer. Do not remove the entire supernatant, as this will lead to loss of nuclei.
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.
Note
CRITICAL: Be careful not to disturb the nuclei pellet.
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.
Note
For 2 yr old mice - pellet was resuspended in 750µL NRB.
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.
Note
The nuclei concentration will decrease by 30%–40% following filtration.
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.
Note
If nuclei are diluted while counting, multiply the equation for calculating the concentration of nuclei with the dilution factor.
Note
CRITICAL: Verify that nuclei are mostly intact and not clumping (Figures 3A and 3B). Note,
that intact nuclei isolated from adipose tissues have a heterogenic morphology.
Citations
Van Hauwaert EL, Gammelmark E, Sárvári AK, Larsen L, Nielsen R, Madsen JGS, Mandrup S. Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics.
Isolation of nuclei from mouse white adipose tissues for single-nucleus genomics.pdf2.6MB