TITLE: Sample preparation for single nuclei sequencing of brain
AUTHORS: Anna Martinez Casals, Nicholas Mitsios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Sample preparation for single nuclei sequencing using fresh frozen tissue from human postmortem brain. The protocol is a combination of an already existing and published protocol in Nature protocols </span><span style = "font-style:italic;">Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons</span><span> and the 10x Genomics handbook </span><span style = "font-style:italic;">Sample preparation, isolation of nuclei for single nuclei RNA sequencing</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [Nuclei isolation]
Place a Petri dish on dry ice and transfer the tissue there, cut it using a chilled scalpel. Transfer the tissue into the precooled Dounce homogenizer and keep the homogenizer on ice during the rest of the protocol ( to reduce heat caused by friction).
?. [Nuclei isolation]
Homogenize the tissue with five strokes of the loose pestle, followed by 10–15 strokes of the tight pestle.
?. [Nuclei isolation]
Filter the homogenate through a BD Falcon tube with a cell strainer cap.
?. [Nuclei isolation]
Transfer the volume to a chilled 1.5 ml eppendorf and centrifuge at 1000 rcf forat .
?. [Nuclei isolation]
Remove the supernatant without disrupting the nuclei pellet.
?. [Myelin removal]
Add nuclei wash and resuspension buffer to the pelleted nuclei and, using a regular-bore pipette tip, gently pipette mix 8-10 times.
1800 µl
?. [Myelin removal]
Add Myelin Removal Beads II to the resuspended nuclei. Mix with a wide-bore pipette tip (no vortex). Incubate for at .
200 µl
?. [Myelin removal]
Meanwhile, place LS columns in the magnetic field of a MACS separator each with  LS Column Calibration Buffer to rinse the columns.
3 ml
?. [Myelin removal]
Using a 10 ml serological pipette, dilute the nuclei suspension with Nuclei Wash and Resuspension buffer, gently pipette mix 5 times.
10 ml
?. [Myelin removal]
Centrifuge the nuclei at 500 rcf for at .
?. [Myelin removal]
Remove the supernatant without disrupting the nuclei pellet and add Nuclei Wash and Resuspension buffer.
2 ml
?. [Myelin removal]
Apply  nuclei suspension in each column.
1 ml
?. [Myelin removal]
Wash each of the columns withNuclei Wash and Resuspension buffer
1 ml
[once more.]
?. [Myelin removal]
Collect the effluent into two 5ml Eppendorf tubes.
?. [Myelin removal]
Centrifuge the two vials with nuclei at 500 rcf for at .
?. [Myelin removal]
Remove the supernatant from the two vials without disrupting the nuclei pellet and add Nuclei Wash and Resuspension buffer.
500 µl
?. [Myelin removal]
Pool both resuspended nuclei in a new pre-chilled 1.5 ml  Eppendorf.
?. [Density gradient centrifugation]
Centrifuge at 1000 rcf for at .
?. [Density gradient centrifugation]
Aspirate the supernatant (~1,000 μl) and gently resuspend the pellet in 250 μl of homogenization buffer.
?. [Density gradient centrifugation]
Meanwhile,  prepare the iodixanol dilution mix (IDM) and iodixanol dilutions to place them on ice.
?. [Density gradient centrifugation]
Strain the mixture through a BD Falcon tube with a cell strainer cap to remove any remaining aggregates, and place it on ice.
?. [Density gradient centrifugation]
Gently mix the nuclei with 250 μl of 50% (vol/vol) iodixanol, and, to a new Eppendorf tube, add 500 μl of 29% iodixanol. Slowly layer 500 μl of the nuclei mixture over the 29% iodixanol.
?. [Density gradient centrifugation]
Centrifuge at 13.500 rcf for at .
?. [Density gradient centrifugation]
Remove and discard the supernatant without disrupting the nuclei pellet.
?. [Density gradient centrifugation]
Resuspend the final pellet in  Nuclei Wash and Resuspension buffer. The sample is ready for QC-nuclei counting.
60 µl