Sep 22, 2020

Public workspaceNuclei Isolation for Single-Nuclei RNA sequencing

DOI
dx.doi.org/10.17504/protocols.io.bkacksaw
  • Danh Truong1,
  • Salah-Eddine Lamhamedi-Cherradi1,
  • Joseph A. Ludwig1
  • 1MD Anderson Cancer Center
  • Human Cell Atlas Method Development Community
  • Ludwig Lab
Danh Truong
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DOI: dx.doi.org/10.17504/protocols.io.bkacksaw
Protocol Citation: Danh Truong, Salah-Eddine Lamhamedi-Cherradi, Joseph A. Ludwig 2020. Nuclei Isolation for Single-Nuclei RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bkacksaw
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: August 25, 2020
Last Modified: September 22, 2020
Protocol Integer ID: 40996
Keywords: single-cell, single-nuclei, nuclei isolation, fatty tissue, adipose tissue, liposarcoma, sarcoma, PDX,
Abstract
We developed this protocol while trying to isolate single-nuclei from frozen liposarcoma tissue. We tested this protocol on a variety of PDX's. For fattier tissues, we use a modified protocol based on a different kit. Thanks to Dr. Luciano Martelotto for his assistance and tips on the first section.

This protocol is based on prior work: ‘Frankenstein’ protocol, Nuclei Isolation Kit: Nuclei EZ Prep, and Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes.
Guidelines
Refer to Isolation of Nuclei for Single Cell RNA Sequencing from 10x genomics for additional information.

For filtering the nuclei, we don't use use Flowmi filters as we find that some of the tips do not fit properly into the filters and I lost some samples due to this. I prefer to use the pluriStrainer Mini.

For nuclei sequencing, since nuclei have lower RNA content compared to whole cell, protocols could increase the number of cDNA amplification cycles by 1 or 2 cycles, as suggested by 10x genomics.
Materials
MATERIALS
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571
ReagentProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
ReagentRNase-Free Disposable Pellet Pestles, With tubeThermo FisherCatalog #12141368
ReagentRNase InhibitorThermo FisherCatalog #N8080119
ReagentNuclei Isolation Kit: Nuclei EZ PrepSigma AldrichCatalog #NUC-101
ReagentPluriStrainer Mini 40µmpluriSelectCatalog #43-10040-60
ReagentMinute Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured AdipocytesFisher ScientificCatalog #AN-029
ReagentBioVortexer MixerFisher ScientificCatalog #50-153-2378
ReagentpluriStrainer Mini 70 UMFisher ScientificCatalog #NC1444112
STEP MATERIALS
ReagentpluriStrainer 40 µmpluriSelectCatalog #43-50040
ReagentNuclei EZ lysis buffer SigmaCatalog #EZ PREP NUC-101
ReagentMinute Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured AdipocytesFisher ScientificCatalog #AN-029
ReagentBioVortexer MixerFisher ScientificCatalog #50-153-2378
ReagentRNase-Free Disposable Pellet Pestles, With tubeThermo FisherCatalog #12141368
ReagentBioVortexer MixerFisher ScientificCatalog #50-153-2378
ReagentRNase-Free Disposable Pellet Pestles, With tubeThermo FisherCatalog #12141368
ReagentpluriStrainer Mini 70 UMFisher ScientificCatalog #NC1444112
Consumables:
  • Scalpels
  • 1.5 mL tubes
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • PBS, sterile
Protocol materials
ReagentNuclei Isolation Kit: Nuclei EZ PrepMerck MilliporeSigma (Sigma-Aldrich)Catalog #NUC-101
Materials
ReagentBioVortexer MixerFisher ScientificCatalog #50-153-2378
In Materials, Materials, Materials and 2 steps
ReagentpluriStrainer Mini 70 UMFisher ScientificCatalog #NC1444112
In Materials, Materials, Step 6
ReagentProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
Materials
ReagentNuclei EZ lysis buffer Merck MilliporeSigma (Sigma-Aldrich)Catalog #EZ PREP NUC-101
Materials, Step 3
ReagentRNase InhibitorThermo FisherCatalog #N8080119
Materials
ReagentRNase-Free Disposable Pellet Pestles, With tubeThermo FisherCatalog #12141368
In Materials, Materials, Materials and 2 steps
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571
Materials
ReagentpluriStrainer 40 µmpluriSelectCatalog #43-50040
Materials, Step 18
ReagentMinute Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured AdipocytesFisher ScientificCatalog #AN-029
In Materials, Materials, Step 23
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020
Materials
ReagentPluriStrainer Mini 40µmpluriSelectCatalog #43-10040-60
Materials
Before start
All solutions should be kept atTemperature4 °C or onTemperatureOn ice prior to use.

Turn on benchtop centrifuge and keep atTemperature4 °C

Prepare Nuclei Wash and Resuspension Buffer (Temperature4 °C )
  • 1X PBS with 1.0% BSA and 0.2U/μl RNase Inhibitor

Nuclei Isolation for Fatty tissues
  • use a thermometer to make sure freezer is aboutTemperature-20 °C
  • Chill plastic consumables in ice before use
Nuclei Isolation (most tissues)
Nuclei Isolation (most tissues)
25m
25m
This protocol should work for most tissues. If the tissue that you are working is fatty or contains fat, including adipose or brain, skip to Nuclei Isolation for Fatty tissues section.
Critical
Use a scalpel and mince tissue on ice. Mincing tissue will improve nuclei isolation efficiency.
AddAmount500 µL of EZ Lysis Buffer to tissue in a 1.5 mL microcentrifuge tube.

ReagentNuclei EZ lysis buffer Sigma AldrichCatalog #EZ PREP NUC-101


Homogenize tissue using plastic microcentrifuge pestles. A Chemglass Life SciencesSupplier BioVortexer Mixer [Fisher Scientific: 50-153-2378] can be used. This should be done in ice but you can take it out for visual inspection. The timing will be based on tissue type and size.

ReagentBioVortexer MixerSigma AldrichCatalog #50-153-2378

ReagentRNase-Free Disposable Pellet Pestles, With tubeSigma AldrichCatalog #12141368

45s
AddAmount1 mL of EZ Lysis Buffer to the 1.5 mL microcentrifuge tube and incubateTemperatureOn ice forDuration00:05:00 . This time can be modified depending on your tissue type and size.

5m
Filter the solution using pluriStrainer Mini 70 μm [Fisher Scientific: NC1444112] into a new 1.5 mL microcentrifuge tube.

ReagentpluriStrainer Mini 70 UMSigma AldrichCatalog #NC1444112

Centrifuge the solution atCentrifigation500 x g, 4°C, 00:05:00 .

5m
AddAmount1 mL of EZ Lysis Buffer to the 1.5 mL microcentrifuge tube and incubateTemperatureOn ice forDuration00:05:00 .

Centrifuge the solution atCentrifigation500 x g, 4°C, 00:05:00 .
Remove the supernatant while being careful not to disturb the pellet. If you can not see the pellet, it is advisable to leave behindAmount50 µL .

Critical
Washing
Washing
15m
15m
Carefully addAmount0.5 mL of the Nuclei Wash and Resuspension Buffer and incubate forDuration00:05:00 without resuspending.

AddAmount0.5 mL of the Nuclei Wash and Resuspension Buffer and resuspend the nuclei.
Centrifuge the solution atCentrifigation500 x g, 4°C, 00:05:00 .
5m
Aspirate the supernatant while being careful not to disturb the pellet and leave behindAmount50 µL .
AddAmount1 mL of Nuclei Wash and Resuspension Buffer.
Centrifuge the solution atCentrifigation500 x g, 4°C, 00:05:00 .
5m
Aspirate the supernatant and resuspend the nuclei inAmount500 µL of Nuclei Wash and Resuspension Buffer supplemented withConcentration10 µg/µL DAPI.

Filter nuclei with a pluriStrainer Mini 40 μm [Fisher Scientific: NC1469671].
ReagentpluriStrainer 40 µmSigma AldrichCatalog #43-50040

Visual Inspection and Sorting
Visual Inspection and Sorting
1h
1h
Visually inspect nuclei integrity under a microscope and count the number of nuclei with a cell counter. This is important to check before continuing to see if the protocol was successuful.
Image of isolated nuclei. There is significant debris prior to sorting.

Imaging
Critical
Nuclei should be sorted as there will be significant debris after dissociation.
Report of DAPI sorted particles

45m
Analyze
Nuclei can be visualized after sort to confirm success.


Image of isolated nuclei after sorting.



Imaging
After sorting, adjust concentration of nuclei to 700 nuclei/μl if needed. Continue with 10x Genomics Single Cell Protocol.
Nuclei Isolation for Fatty tissues
Nuclei Isolation for Fatty tissues
10m
10m
For fatty tissues, including brain and adipose, the lipids can interfere with the collection. This is a different protocol that can assist in enhancing nuclei isolation efficiency. We will use a modified protocol from the Minute Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured Adipocytes kit.

ReagentMinute Nuclei and Cytosol Isolation Kit for Adipose Tissues/Cultured AdipocytesSigma AldrichCatalog #AN-029

Critical
Tissue weight should range fromAmount120 mg toAmount150 mg . Mince tissue using a scalpel and place inside a 1.5 mL microcentrifuge tube.

AddAmount600 µL of N/C buffer to tissue in a 1.5 mL microcentrifuge tube.
Homogenize tissue using plastic microcentrifuge pestles. A Chemglass Life SciencesSupplier BioVortexer Mixer [Fisher Scientific: 50-153-2378] can be used. This should be done in ice but you can take it out for visual inspection. The timing will be based on tissue type and size.

ReagentBioVortexer MixerSigma AldrichCatalog #50-153-2378

ReagentRNase-Free Disposable Pellet Pestles, With tubeSigma AldrichCatalog #12141368

Filter Nuclei and Lipids
Filter Nuclei and Lipids
30m
30m
Place the filter catridge that comes with the kit onto the collection tube. Pour the homognized tissue and liquid into the filter. Pipette any remaining liquid into the filter.
Incubate the filter cartridge with the cap open in a freezer atTemperature-20 °C for Duration00:20:00

20m
The timing can be optimized if you do not have a thermometer. AddAmount0.5 mL of ddH2O in a 1.5 mL microcentrifuge tube and incubate in the freezer until the liquid freezes. This is the time that can be used.

Place the tube with cap open in a microcentrifuge. Centrifuge atCentrifigation500 x g, 4°C, 00:03:00 . Increase toCentrifigation1000 x g, 4°C, 00:01:00 if there is some liquid retention. You will see fat stuck on top of filter. The supernatant below will contain the nuclei.

Discard the filter and close the microcentrifuge tube. Vortex briefly to mix up the nuclei. Make sure you know where the pellet will end up based on the placement inside the centrifuge. The nuclei pellet will be white or almost invisibible. Centrifuge atCentrifigation1000 x g, 4°C, 00:04:00 .
The supernatant can be saved if needed. It is the cytosolic fraction. I did not have a visible nuclei pellet but I marked where the pellet would be. Carefully aspirate the liquid and leave some behind if needed. Resuspend nuclei inAmount50 µL Nuclei Wash and Resuspension Buffer.



Transfer nuclei to a clean microcentrifuge tube to avoid lipid contamination leftover on the tube walls.
The nuclei can visualized using trypan blue. You will notice lipid contamination still, which can be removed using FACS.

Isolated nuclei from fatty tissue.

Imaging
While both protocols will result in many lipid droplets in addition to the nuclei, the fatty tissue protocol will have a higher nuclei count overall.

Left graph shows results from the standard protocol. Right graph shows the results from the fatty tissue protocol.

Analyze
Go togo to step #22 for sorting prior to 10x Genomics Single Cell protocol.
Cryopreservation
Cryopreservation
1h 30m
1h 30m
AddAmount900 µL RNALater toAmount100 µL nuclei in Nuclei Wash and Resuspension Buffer, incubate atTemperature4 °C forDuration01:00:00 then transfer toTemperature-80 °C
1h
To remove RNALater, centrifuge nuclei atCentrifigation4000 x g, 4°C, 00:10:00 . Aspirate RNALater and resuspend in Nuclei Wash and Resuspension Buffer
10m