TITLE: Nuclei Isolation for Single-Nuclei RNA sequencing
AUTHORS: Danh Truong,  Salah-Eddine Lamhamedi-Cherradi, Joseph A. Ludwig

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block"><span>This protocol is based on prior work: </span><a href="https://www.protocols.io/view/frankenstein-protocol-for-nuclei-isolation-from-f-3fkgjkw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">‘Frankenstein’ protocol</span></a><span>, </span><a href="https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/nuc101bul.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Nuclei Isolation Kit: Nuclei EZ Prep</span></a><span>, and </span><a href="https://cdn.shopify.com/s/files/1/0063/4575/2687/files/22-AT-022_v5.pdf?321" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes</span></a><span>.</span></div></div>

[STEPS]
?. [Nuclei Isolation (most tissues)]
Use a scalpel and mince tissue on ice. Mincing tissue will improve nuclei isolation efficiency.
?. [Nuclei Isolation (most tissues)]
Addof EZ Lysis Buffer to tissue in a 1.5 mL microcentrifuge tube.
500 µl
?. [Nuclei Isolation (most tissues)]
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.
?. [Nuclei Isolation (most tissues)]
Addof EZ Lysis Buffer to the 1.5 mL microcentrifuge tube and incubatefor. This time can be modified depending on your tissue type and size.
1 mL
on ice
?. [Nuclei Isolation (most tissues)]
Filter the solution using pluriStrainer Mini 70 μm [Fisher Scientific: NC1444112] into a new 1.5 mL microcentrifuge tube.
?. [Nuclei Isolation (most tissues)]
Centrifuge the solution at .
Centrifuge: 500 34, 5 min, 4 10
?. [Nuclei Isolation (most tissues)]
Remove the supernatant while being careful not to disturb the pellet. If you can not see the pellet, it is advisable to leave behind .
50 µl
?. [Washing]
Carefully add of the Nuclei Wash and Resuspension Buffer and incubate for without resuspending.
0.5 mL
?. [Washing]
Centrifuge the solution at.
Centrifuge: 500 34, 5 min, 4 10
?. [Washing]
Aspirate the supernatant and resuspend the nuclei inof Nuclei Wash and Resuspension Buffer supplemented withDAPI.
500 µl
?. [Washing]
Aspirate the supernatant while being careful not to disturb the pellet and leave behind .
50 µl
?. [Washing]
Addof Nuclei Wash and Resuspension Buffer.
1 mL
?. [Washing]
Centrifuge the solution at.
Centrifuge: 500 34, 5 min, 4 10
?. [Washing]
Filter nuclei with a pluriStrainer Mini 40 μm [Fisher Scientific: NC1469671].
?. [Visual Inspection and Sorting]
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.
?. [Visual Inspection and Sorting]
Nuclei should be sorted as there will be significant debris after dissociation.
?. [Visual Inspection and Sorting]
After sorting, adjust concentration of nuclei to 700 nuclei/μl if needed. Continue with 10x Genomics Single Cell Protocol.
?. [Nuclei Isolation for Fatty tissues]
Tissue weight should range fromto. Mince tissue using a scalpel and place inside a 1.5 mL microcentrifuge tube.
120 mg
150 mg
?. [Visual Inspection and Sorting]
Nuclei can be visualized after sort to confirm success.
?. [Nuclei Isolation for Fatty tissues]
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.
?. [Nuclei Isolation for Fatty tissues]
Addof N/C buffer to tissue in a 1.5 mL microcentrifuge tube.
600 µl
?. [Filter Nuclei and Lipids]
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.
?. [Nuclei Isolation for Fatty tissues]
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.
?. [Filter Nuclei and Lipids]
Incubate the filter cartridge with the cap open in a freezer atfor
-20 °C
?. [Filter Nuclei and Lipids]
Place the tube with cap open in a microcentrifuge. Centrifuge at. Increase toif there is some liquid retention. You will see fat stuck on top of filter. The supernatant below will contain the nuclei.
Centrifuge: 500 34, 3 min, 4 10
Centrifuge: 1000 34, 1 min, 4 10
?. [Filter Nuclei and Lipids]
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 at.
Centrifuge: 1000 34, 4 min, 4 10
?. [Filter Nuclei and Lipids]
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 inNuclei Wash and Resuspension Buffer.
50 µl
?. [Filter Nuclei and Lipids]
The timing can be optimized if you do not have a thermometer. Addof 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.
0.5 mL
?. [Filter Nuclei and Lipids]
for sorting prior to 10x Genomics Single Cell protocol.
?. [Nuclei Isolation (most tissues)]
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.
?. [Cryopreservation]
AddRNALater tonuclei in Nuclei Wash and Resuspension Buffer, incubate atforthen transfer to
900 µl
100 µl
4 °C
-80 °C
?. [Cryopreservation]
To remove RNALater, centrifuge nuclei at. Aspirate RNALater and resuspend in Nuclei Wash and Resuspension Buffer
Centrifuge: 4000 34, 10 min, 4 10
?. [Filter Nuclei and Lipids]
The nuclei can visualized using trypan blue. You will notice lipid contamination still, which can be removed using FACS.
?. [Filter Nuclei and Lipids]
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.
?. [Nuclei Isolation (most tissues)]
Addof EZ Lysis Buffer to the 1.5 mL microcentrifuge tube and incubatefor.
1 mL
on ice
?. [Nuclei Isolation (most tissues)]
Centrifuge the solution at .
Centrifuge: 500 34, 5 min, 4 10
?. [Washing]
Addof the Nuclei Wash and Resuspension Buffer and resuspend the nuclei.
0.5 mL
?. [Filter Nuclei and Lipids]
Transfer nuclei to a clean microcentrifuge tube to avoid lipid contamination leftover on the tube walls.