TITLE: Frozen tissue dissociation for single-nucleus RNA-Seq
AUTHORS: Linas Mazutis, Ignas Masilionis, Ojasvi Chaudhary

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol described here relies on mechanical dissociation of frozen lung tissue (human or mouse), filtering and washing the nuclei suspension in SCC buffer followed by the FACS sorting. The resulting nuclei suspension can be processed on scRNA-Seq platform of choice, 10X Chromium, inDrops or other. The protocol has been validated on frozen human tissues of lung, breast pancreas and kidney as well as frozen mouse tissues of brain, lung, pancreas. </div></div>

[STEPS]
?. [Tissue grinding]
Prepare the laminar hood and the grinding glassware:Clean the surface and glassware thoroughly with DRNAse Free Reagent Spray followed by 70% ethanol. Bring the ice bucket in the cell culture hood and spray it with DRNAse Free Reagent.Place fully assembled Dounce homogenizer on ice and to let it cool.Bring sterile blade in the hood.
?. [Tissue grinding]
Prepare a frozen tissue:
?. [Tissue grinding]
Prepare Lysis buffer:Transfer  in 1.5 mL tube, add  of DEPC and vortex vigorously until solution becomes cloudy and homogenous.
[Lysis Buffer]
1 µl
DEPC is not soluble in water and it will form layer of droplets on top. Therefore, mix vigorously to disperse DEPC droplets.
?. [Tissue grinding]
Grind the tissue:Place the small piece(s) of frozen tissue in 2 mL grinding glass tube.Using a large clearance pestle (Tube A) for the initial sample grinding gently move pestle up and down 10-15 times.  Next, use a small clearance pestle (Tube B) and grind tissue further by movinig up and down for 10-15 times.Tissue should be completely minced and homogenized.  Keep the sample in a glass tube on ice.Next, use a small clearance pestle (Tube B) and grind tissue further by movinig up and down for 10-15 times.Tissue should be completely minced and homogenized.  Keep the sample in a glass tube on ice.
Do not remove pestle out of the liquid while grinding the tissue, avoid creating bubbles, which may form if pestle is being moved too quickly.
Certain tissue types might be elastic and hard to break down completely. In such case move to next step.
?. [Tissue grinding]
Record experimental details:
?. [Straining/washing of single-nuclei suspension]
IMPORTANT: keep nuclei suspension cool at all times.NOTE: For centrifugation use a swinging bucket centrifugure and 1.5 ml (or 2.0 ml) Protein LoBind Eppendorf tubes placed inside the 50 mL Falcon tube.
?. [Straining/washing of single-nuclei suspension]
Centrifugation:Spin down the tube in a swinging bucket centrifuge at .
Centrifuge: 500 34, 5 min, 4 10
?. [Straining/washing of single-nuclei suspension]
Centrifugation:Spin down the dissociated tissue suspension in a swinging bucket centrifuge at .There should be a clearly visible pellet. If you don't see one, check the settings on your centrifuge.Remove and save supernatant from the centrifuged nuclei pellet. Do not disturb the pellet, leave ~ 10-20 uL of supernatant on top.
Centrifuge: 500 34, 5 min, 4 10
?. [Straining/washing of single-nuclei suspension]
Straining:Place 5 mL Falcon Round-bottom tube (typically used for FACS) with blue Snap Cap strainer on ice.Strain the nuclei suspension through 35 µm Cell Strainer Snap Cap and collect flow through fraction into the tube. Transfer the filtered solution to 1.5 mL Protein LoBind Eppendorf tube, keep on ice.
If it is difficult to aspirate the nuclei suspension by pipette another option is to pour all suspension out of 2 mL homogenizer tube directly onto the blue cap. However, care must be taken to not introduce any ice that might be stuck on the outside walls of a homogenizer tube.
?. [Straining/washing of single-nuclei suspension]
Resuspension:Re-suspend the pellet in  using 1000 µL pipette.
[Nuclei Wash Buffer]
?. [Straining/washing of single-nuclei suspension]
Resuspension and straining:Re-suspend the nuclei pellet in , mix well using 1000 µL pipette until suspension becomes homogenious. The volume of buffer at this step can be adjusted as needed (e.g. 0.5 ml).
[Nuclei Wash Buffer]
?. [Straining/washing of single-nuclei suspension]
Nuclei evaluation under BF/FL microscope:Mix  with   and  .Inspect nuclei suspension under the bright-field and fluorescence microscope. Record the images (Figure 1).Approximately 106- 107 nuclei per 1 ml should be expected.
[nuclei suspension]
[100X DAPI dye]
[0.4% Trypan Blue dye]
When evaluating sample under the bright field there should be a lot of dark debris visible (Figure 1) making it hard to count the species. However, i the blue (DAPI) channel the nuclei should be clearly visible.
?. [FACS]
Prepare the unstained nuclei sample: Take   and transfer to another 5 mL Falcon tube. Add  .Label the tube as “reference” which will be used as a unstained population reference for FACS.Keep on ice
[nuclei suspension]
[Nuclei Resuspension Buffer]
?. [FACS]
Prepare the DAPI-stained nuclei sample: Take remaining,  and stain with   by mixing the suspension with 1 ml pipette.
[nuclei suspension]
[100X DAPI dye ]
?. [FACS]
Prepare the FACS collection tube: Add  into 5 mL Falcon tube and swirl buffer all around to wet the inner walls.
[Nuclei Resuspension Buffer]
?. [FACS]
Sort DAPI positive nuclei on FACSRun the "reference" sample (from Step 13) and record the scatter plot.Run DAPI-positive nuclei suspenion (from Step 14),  indetify the fluorescent population and record the scatter plot. Using the collection tube preapred in Step 15, sort the DAPI-postiive population into the collection tube The example of sorting gates (P4) is provided below:
?. [Post-FACS]
Concentrate the nucleiConcentrate nuclei by transferring FACS sorted suspension in 1.5 mL Protein LoBind tube and spinning in a swinging bucket centrifuge at .
Centrifuge: 600 34, 5 min, 4 10
Using FACS counts aim to reach a dilution of 2000 nuclei/ul.
?. [Post-FACS]
Count the nucleiAfter centrifugation carefully aspirate the supernatant leaving the desirable amount of suspension (e.g. 200 µl) Carefully disperse the nuclei by mixing suspension with a 200 µl pipette.Mixi    with   and   and count the nuclei on Countess II instrument. Expected results are shown in Figure 3 and Figure 4.Mixi   with  and  and count the nuclei on Countess II instrument.
[Nuclei Suspension]
[100X DAPI dye]
[0.4% Trypan Blue dye]
?. [Single nucleus RNA-Seq]
Control experiment:If frozen tissue dissociation is being conducted for the first time perform bulk cDNA synthesis and evaluate the cDNA yields and profile. Otherwise, proceed to the reverse transcription reaction using the platform and method of your choice.
?. [Single nucleus RNA-Seq]
Barcoding, library preparation and sequencingFollow 10X genomics protocol when using Chromium instrument 10X genomics protocol (v3.1 Chemistry)Follow inDrops protocol when using home-built inDrops plaform inDrops protocol
?. [Sequencing]
Sequence the DNA libraryWe use NovaSeq 6000 instrument to sequence the final DNA libraries using pair-end sequencing option, R1 read - 26 cycles, R2 read - 70 cycles, and index read - 8 cycles, aiming for ∼100 million reads per ~5,000 single-nuclei.
The expected results are :
Frozen Human Lung - SCLC
Frozen Human Lung - cSCLC