TITLE: Extraction of Nuclei from Brain Tissue
AUTHORS: Laura Bortolin, Melissa Goldman, Steven McCarroll

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol enables rapid isolation of intact nuclei from brain tissue, including highly myelinated adult brain tissue.  We have used it successfully to extract nuclei for single-nucleus RNA-seq from human, mouse, marmoset, and macaque brain.  </div></div>

[STEPS]
?. Retrieve dissection tray from -20°C, and the glass slide and razor blade from 4°C.
?. Place the glass slide, razor blade, and ruler on the dissection tray and perform all cutting on slide to minimize damage to, and contamination of, the tray.
It is helpful to put the dissecting tray on ice to keep it cold and prevent it from moving.
?. Place the frozen sample on the glass slide and shave off the frosty/freezer-burned tissue from the face you want to sample from, and discard. This is damaged and not worth extracting. Then, moving rapidly but carefully, shave off enough sample to work with.
?. Return the tissue to -80°C (or put on dry ice as a temporary hold).
?. Rapidly mince the shaved sample with the same razor blade that was used to shave the tissue in step 3 and scrape into the first well of a 6-well plate that contains 4 mL of cold extraction buffer. Pipette up and down 20 times.
?. Commence 10-minute incubation, pipetting up and down 20x every ~2.5 minutes with a 1000 uL pipette.
4 °C
?. Pull the sample into the chilled needle/syringe and express (expel) into the second well of the 6-well plate.
?. Repeat this, pulling the sample into the same syringe and expressing it into the third well of a 6-well plate.
Note that one may simultaneously process 2 distinct samples in the top and bottom 3 wells of a 6-well plate.
?. Wet the 20 µm filter with 200 uL cold WB, then pull the sample through the filter using gentle vacuum. Then pass 2 mL of cold WB through the filter as a wash step.
Try to avoid frothing (bubbles).
?. Optional Step: Wet the 5 µm filter with 200 uL WB and put the sample through that filter, using gentle vacuum. Try to avoid frothing. Then pass 2 mL of cold WB through the filter as a wash step.
Using the 5 µm filter may not be necessary if you are working with a sample that has a low amount of debris/white matter, or if you do not want to risk filtering out some of the larger nuclei (although most of the larger nuclei will still squeeze through the 5 µm filter with the vacuum).
?. Optional Step: Remove 10 uL of nuclei and mix with 10 mL of the diluted Hoechst stain and view using the brightfield and DAPI filters on the fluorescent scope.  Photograph both brightfield and DAPI-excited nuclei.
This is your baseline extraction and it’s a check to see if you have nuclei (and if it is worth proceeding with the prep).  You will still see debris, despite the filtration steps.  The subsequent spin will remove much of the debris, though some will still pellet with the nuclei.
It is helpful to have a second person perform this step while you continue with step 12.
Once you are familiar with extracting nuclei from a particular type of tissue and are consistently getting the expected nuclei yields, this baseline check step can be skipped.
?. Add chilled WB to the filtered nuclei to a total volume of just under 50 mL.
?. Divide these diluted nuclei among four 50 mL conical tubes (~14 mL per tube).
Don’t use 15 mL tubes as the nuclei do not “travel” the longer distance well.
?. Carefully remove as much of the supernatant as possible without disturbing the pellet.
The pellet is hard-to-impossible to see.  The cleaner the prep, the less visible the pellet is.
?. Resuspend the pellet in ~1 mL of WB, serially combining the pellets from all 4 tubes.
You will end up with ~1.5 - 2 mLs of extracted nuclei (the 1 mL you added plus the remaining WB that you had to leave in the tube to not disturb the pellet).
?. Obtain nuclei counts, calculate concentration, and dilute to the desired concentration using DB.
For Nuclear Drop-seq, we typically dilute to 176 nuclei/uL.
?. Add 10 mL nuclei + 10 mL diluted Hoechst stain and visualize with the fluorescent scope, getting images of brightfield and DAPI-excited nuclei.
It is normal to see some debris but you should see less than what you saw in the baseline visualizations from step 11.
?. Spin at 500 rcf for marmoset and human nuclei (600 rcf for mouse nuclei) for 10 minutes at 4°C.
4 °C