TITLE: Nuclear prep and FACS for 10x Genomics Chromium single-nucleus sequencing
AUTHORS: Matt Keefe

[DESCRIPTION]
Protocol is used for nuclear isolation and single cell RNA sequencing in the Nowakowski lab. 

[STEPS]
SECTION: Introduction
1. Nuclear prep and FACS for 10x Genomics Chromium single-nucleus sequencing
(protocol version 1.3.2, J. Brown, Arlotta lab)
SECTION: Introduction
2. Partially from Habib et al., Massively parallel single-nucleus rna-seq with dronc-seq, Nature Methods 2017. (With additional commentary by Eugene Drokhlyansky and Ehsan Habibi, Regev lab. Per Gene, this method for nuclear extraction works best. Per Ehsan, this method works very well for brain)
SECTION: Reagents
3. EZ Lysis Buffer from Nuclei EZ Prep kit (Sigma, Cat # NUC-101)
        ensure that the lysis buffer is the version designed for cell culture, and not the version designed for tissue
2ml glass dounce tissue grinder (e.g., Sigma, Cat # D8938) 
        if using a different product, ensure pestles have ball end and not U-end, or they will damage the nuclei
35-μm cell strainer tube (e.g., Corning, Cat # 352235)
1ml pipettor and pipette tips (note: 2ml serological pipettes do not fit in the dounce)

Nuclei Suspension Buffer (NSB):
1 × RNAse-free molecular-biology grade PBS
0.1% molecular-biology grade BSA (100 μg/ml) (e.g., NEB B9000S)
0.2U/μl RNase inhibitor (e.g., Clontech, Cat # 2313A)
For 5 ml:
5 ml PBS
25 μl NEB BSA (@ 20 mg/ml)
25 μl RNase inhibitor (@ 40U/ul)
SECTION: Tissue harvesting and storage
4. Dissect tissue and place in 1.5 mL microcentrifuge tube, transferring as little media as possible. If necessary, cut tissue samples into pieces <0.5 cm first. Flash-freeze on dry ice and store at ‑80oC. 
SECTION: Tissue harvesting and storage
5. Dissect the region of interest, place on the inside wall of a 1.5 mL tube, make sure it doesn't slide down, and freeze tube in dry ice. The sliding part is to make sure there is no excess liquid on the tissue. You don't want to pat dry, but you don't want tissue to freeze in any liquid.
SECTION: Nuclear prep
6. Note: Maintain RNAse-free conditions throughout. Keep nuclei ice-cold at all times. Resuspend and mix gently to avoid excessive damage.
SECTION: Nuclear prep
7. Preparation
SECTION: Nuclear prep
7.1. Wash dounce and pestles with 100% EtOH, followed by RNAse Zap. Wash in 2-3 rounds of RNAse-free water (in 50 ml conical), and rinse with EZ Lysis Buffer.
SECTION: Nuclear prep
7.2. Chill dounce and pestles on ice (pestles in a 50 ml conical tube).
SECTION: Nuclear prep
7.3. Prepare ~5 ml Nuclei Suspension Buffer per sample (plus ~1-2 ml extra if doing FACS) and place on ice.
SECTION: Nuclear prep
7.4. If you are using the Vybrant DyeCycle Ruby stain, make ~1 ml NSB & Ruby (1:500) per sample (or a bit extra if you think you might need to dilute the sample). Add an additional 5 μl / ml RNAse inhibitor to this solution (final 0.4U/μl), because the nuclei will be sitting in it for a while. Hold on ice.

The Ruby is provided in a DMSO solution and is not very soluble in cold media; if necessary, dilute it 1/10 in a small amount of room-temperature NSB, then 1/50 into the cold NSB (1:500 final).
SECTION: Nuclear prep
7.5. Prepare ~100 μl of rich-NSB to sort the cells into: 100 μl NSB (without Ruby) with 5 μl extra RNAse inhibitor (final 2U/μl) and 5μl extra BSA (final 1%). Hold on ice.
SECTION: Nuclear prep
7.6. Plan to sort into a 96-well plate; pick one well per sample and coat the entire inside with ~20 μl of the NEB BSA (undiluted), then fill with NSB. Place the plate in a clean ziploc bag to transport it to/from the core, and hold on ice. (The BSA coating step reduces adherence and improves recovery.)
SECTION: Nuclear prep
7.7. Before sorting, draw off the BSA in the wells and rinse the well 1-2 times with ~200 μl ice-cold NSB (the basic one), then fill with ~10-20 μl of rich-NSB (based on the volume you expect to collect – try to keep the final post-sort volume under 30 μl for the v2 kit, or 45 μl for the v3 kit). Try to make sure there are no bubbles.
SECTION: Nuclear prep
7.8. Pre-chill centrifuge.
SECTION: Nuclear prep
7.9. lace a 15-ml conical tube on ice for each sample.
SECTION: Nuclear prep
8. Transfer tissue to chilled dounce. Do not allow tissue to thaw. For pieces of mouse cortex under 0.5 cm, it is not necessary to chop the tissue. Immediately add 2 ml ice-cold EZ Lysis Buffer.
SECTION: Nuclear prep
9. Homogenize with 20 strokes of pestle A and 20 strokes with pestle B. Raise and lower slowly, avoiding bubbles. Keep nuclei on ice at all times.
SECTION: Nuclear prep
10. Transfer suspension to chilled conical (from step 2). Rinse dounce with 2 ml ice-cold EZ Lysis Buffer; transfer rinsate to conical and mix gently. Incubate on ice 5 min.
SECTION: Nuclear prep
11. Centrifuge at 500 × g for 5 min at 4°C.
SECTION: Nuclear prep
12. Resuspend in 4 ml ice-cold EZ Lysis Buffer by gentle pipetting and incubate on ice for 5 min.
SECTION: Nuclear prep
13. Centrifuge at 500 × g for 5 min at 4°C
SECTION: Nuclear prep
14. Resuspend in 4 ml ice-cold Nuclei Suspension Buffer.
SECTION: Nuclear prep
15. Centrifuge at 500 × g for 5 min at 4°C.
SECTION: Nuclear prep
16. Resuspend in ~1 ml ice-cold Nuclei Suspension Buffer with Ruby (depending on expected yield). Filter through a 35-μm cell strainer.
SECTION: Nuclear prep
17. For FACS:
As much as possible, the sorted cells should be collected into a small volume (<30 ul final volume for the v2 kit, <45 for v3) to avoid the need to spin down after sorting. We have found that a 100 uM nozzle leads to a larger collection volume than may be desirable (>30ul for ~7000 events); if a smaller nozzle (e.g., 85 uM) gives adequate nuclear quality, it may be preferable.

For 10x:
For the cDNA amplification step, calculate based on ~50-60% recovery and then add 1-2 cycles over the standard recommended number, as nuclei have less RNA than cells