TITLE: HTAPP_NST- Nuclei isolation from frozen tissue
AUTHORS: Eugene Drokhlyansky, Nicholas Van Wittenberghe, Michal Slyper, Julia Waldman, Asa Segerstolpe, Orit Rozenblatt-Rosen, Aviv Regev

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a nuclei isolation method for frozen tissues based on the previously published protocol by </span><a href="https://www.nature.com/articles/s41467-017-00244-w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Gao et al.</span></a><span> and modified by </span><a href="https://www.biorxiv.org/content/10.1101/746743v4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Drokhlyansky et al</span></a><span>. It can be used on both healthy and disease tissues and is compatible with droplet-based single-nucleus RNA-Seq technology </span><a href="https://www.nature.com/articles/s41591-020-0844-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Slyper et al</span></a><span>.</span></div><div class = "text-block"><span>This method is part of a toolbox for processing frozen tissue samples for single-nucleus RNA-Seq, including the NST (this protocol), CST and TST protocols (all available in </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>). We recommend users to test all three protocols on their tissue type of interest and perform side-by-side comparison of the data generated.  </span></div><div class = "text-block"><span>A description of the complete toolbox and guidance for testing and selecting methods from the toolbox for processing other tumors can be found in </span><a href="https://www.nature.com/articles/s41591-020-0844-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Slyper et al</span></a><span>.</span></div><div class = "text-block">For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was tested on metastatic breast cancer, ovarian cancer and pediatric neuroblastoma frozen samples. For these cancer types, however, TST was the protocol of choice. When tested on HTAPP samples, NST, in general, underperformed compared to TST and CST  based on several QC parameters. </div></div>

[STEPS]
?. [Nuclei isolation]
Using forceps, place tissue in the pre-filled well of the 6-well plate on ice (containing the 1 ml of NST; Step 1) and chop tissue for 10 minutes in the buffer using spring scissors.
[On ice]
?. [Nuclei isolation]
With a 1 ml pipette transfer the suspension from the 6-well plate to a 40 µm filter, and filter the entire volume into a 50 ml conical tube.Note: Some small pieces of tissue may be left after 10 minutes of chopping.
[on ice]
?. [Nuclei isolation]
Wash the well with an additional 1 ml of NST buffer and then pass through the same filter.
[NST]
[on ice]
?. [Nuclei isolation]
Wash filter with additional 3 ml of 1X ST buffer. Discard filter.
[ST 1X]
?. [Nuclei isolation]
Transfer total volume (~5 ml) from the 50 ml tube into a clean 15 ml conical tube.
[on ice]
?. [Nuclei isolation]
Centrifuge in a swinging bucket rotor for 5 minutes at 500 g at 4°C. Set stop break to 'soft'.
Centrifuge: 500 34
4 °C
?. [Nuclei isolation]
Carefully take the sample out of centrifuge, and place it on ice. Remove supernatant with a 1 ml pipette and discard.
?. [Nuclei isolation]
Resuspend nuclei pellet in 1X ST buffer (determine the ST volume based on nuclei pellet size - usually around 100-150 µl 1X ST).Note: The volume used for resuspension may vary between tissues and will depend on pellet size and nuclei concentration desired, usually 1000 nuclei / µl. It is better to resuspend initially in a small volume and dilute as needed to avoid an extra centrifugation step to concentrate the nuclei.
?. [Nuclei isolation]
Using a P200 pipette, collect the nuclei suspension and place it on a 35 µm falcon cup filter. Filter the suspension into the 5 ml falcon tube.
?. [Nuclei counting]
Count nuclei using INCYTO C-Chip Neubauer Improved Disposable Hemacytometers and dilute if necessary. The recommended concentration is 1,000 nuclei/µl, but concentrations between 175 and 2,000 nuclei/µl are acceptable. 8,000-10,000 nuclei are typically loaded per channel of a 10x Genomics chip in a volume that should not exceed 43.2 µl for v3.1 chips.Counting Instructions:Load 10 µl of nuclei suspension onto a hemocytometer. Under a microscope, count nuclei that are within the hemocytometer grid (figure below). The full grid contains 9 squares, each of which is 1 mm2. The thickness of liquid in the hemocytometer is 0.1 mm. Consequently, the volume above each of these squares is 0.1 µl and the concentration of nuclei can be calculated as:concentration in nuclei/µl = number of nuclei in 1 mm2 square x 10If the sample has been diluted before loading onto the hemocytometer, make sure to take this dilution into consideration when calculating the sample concentration. For a robust estimate of nuclei concentration, count at least 30 nuclei ideally located in different areas of the grid.Alternatively, nuclei can be stained using DAPI and counted under a fluorescence microscope. In this case, mix 3.3 µl of nuclei suspension with 6.6 µl of DAPI (2.5 µg/µl stock in PBS), load on the hemocytometer, and count as described above. Taking into account the dilution with DAPI solution, the nuclei concentration can be calculated as:concentration in nuclei/µl = number of nuclei in 1 mm2 square x 10 x 3
?. [10x loading]
Load sample on 10x (recommended to load between 8,000-10,000 nuclei per 10x channel).
?. Place all buffers, tubes, and a 6-well plate on ice; pre-fill one well with 1 ml NST buffer. Keep tissue frozen (on dry ice) until begining of processing. Note: If the sample is frozen in OCT, before starting the nuclei isolation protocol cut and discard as much of the frozen OCT surrounding the tissue using a razor and remove the remaining OCT by washing the sample in cold PBS in a 10 cm tissue culture dish (on ice). If needed, using forceps gently assist the removal of the remaining OCT. This should be done on wet ice and as quickly as possible. Quickly after proceed to Step 2. If the sample, however, is an OCT tissue scroll, place the scroll in cold PBS in a tissue culture dish (on ice) and allow for the OCT to dissolve (this should only take a few seconds). Quickly after proceed to Step 2.
[NST]