TITLE: Isolation of single nuclei from solid tissues
AUTHORS: Blue Lake, Kun Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can circumvent the non-uniform or incomplete dissociation of solid tissues into single cells. The isolation of nuclei can also circumvent RNA degradation or any introduction of technical artefacts (such as stress responses) that could be triggered during whole cell dissociation methods. Data generated from single-nucleus genomic assays permits discovery of molecular cell types that can be used to define the overall cellular makeup of a tissue or organ, and ultimately will inform upon adult human tissue atlases.</div></div>

[STEPS]
?. [Prepare Reagents and  Tissue]
Prepare NEB-complete (NEB containing 5 ug/ml DAPI and 0.04 U/ul RNAse Inhibitor)chill on ice  ABC1Final ConcentrationStockVolume (25 ml)220 mM Tris [pH 8]
  320 mM sucrose 
  5 mM CaCl2 
  3 mM MgAc2
  0.1 mM EDTA
  0.1% TritonX-100
  dH201M 
  1M 
  1M 
  1M 
  0.5M 
  10%
  -0.5ml
  8ml
  125µl
  75µl
  5µl
  250µl
  16ml
ABC1Final ConcentrationStockVolume (25 ml)220 mM Tris [pH 8]
  320 mM sucrose 
  5 mM CaCl2 
  3 mM MgAc2
  0.1 mM EDTA
  0.1% TritonX-100
  dH201M 
  1M 
  1M 
  1M 
  0.5M 
  10%
  -0.5ml
  8ml
  125µl
  75µl
  5µl
  250µl
  16ml
For chromatin accessibility assays, include 1:100 dilution of cOmplete™ Protease Inhibitor Cocktail (stock one tablet in 0.5 ml H2O)
?. [Prepare Reagents and  Tissue]
Treat dounce with RNAseZap, rinse with sterile water (if possible: UV treat )
?. [Prepare Reagents and  Tissue]
Transfer vial containing tissue to ice.
For solid tissues (e.g. adult human kidney), 40 µm cryosections can be used with the number of sections dependendent on desired yield and the size and type of tissue. For kidney ~6 cubic mm will give ~150-200K nuclei.
?. [Prepare Reagents and  Tissue]
For sections stored in a stabilizing solution (e.g. RNAlater), wash briefly with PBS and immediately proceed to Step 5 below  ABC1Final ConcStockVolume (50ml)21x PBS 
  1 mM EGTA
  dH2O10x
  0.1M5ml
  50µl
  45ml
ABC1Final ConcStockVolume (50ml)21x PBS 
  1 mM EGTA
  dH2O10x
  0.1M5ml
  50µl
  45ml
?. [Isolate Nuclei]
Add  ice cold NEB buffer to tissue segments
1 ml
?. [Isolate Nuclei]
Cut end off of a p1000 tip to increase bore size using a sterile scalpel, then pipette sections up and down to disperse and dissolve OCT, ~20 times
?. [Isolate Nuclei]
Using a regular p1000 tip, pipette ~10x to further dissociate tissues into manageable sizes. Then transfer to dounce homogenizer
tissue needs to be passable through a p1000 tip easily before proceeding
?. [Isolate Nuclei]
Gently dounce tissue on ice: 5 strokes with pestle A~20 strokes with pestle B (minimize bubble formation)
Increase number of pestle A strokes if the tissue appears too granular before proceeding with pestle B. Number of pestle B strokes used here is dependent on tissue toughness:soft tissues use ~10-15 strokeshard tissues use ~15-20 strokes
Avoid making bubbles
?. [Isolate Nuclei]
Transfer solution to a  tube
15 ml
?. [Isolate Nuclei]
Wash dounce with NEB-complete buffer and add this into the same tube
1 ml
?. [Isolate Nuclei]
Incubate on ice
?. [Isolate Nuclei]
Pass supernatant through 30 uM CellTrics filter to a new  conical tube
15 ml
?. [Isolate Nuclei]
Bring up to  with PBSE
10 ml
?. [Isolate Nuclei]
Pellet nuclei:  at
900 g
4 °C
?. [Isolate Nuclei]
To use nuclei directly for single nucleus assays, proceed to method To use nuclei on a later date, proceed to Step 19
?. [snRNA-Seq methods: nuclei can be stored in RNAlater]
Remove supernatant and resuspend pellet in  -   PBS + 0.1% RNAse Inhibitor
100 µl
1000 µl
Resuspension buffer and volume is dependent on downstream assays and nuclei concentration requirements. 1% BSA can be included here
?. [snRNA-Seq methods: nuclei can be stored in RNAlater]
Add  RNAlater to  nuclei in PBS, incubate at  for  to then transfer to  for 1-2 months
900 µl
100 µl
4 °C
-20 °C
?. [snRNA-Seq methods: nuclei can be stored in RNAlater]
QA/QC: Count nuclei (e.g. BioRad T20 Cell Counter)
?. [snRNA-Seq methods: nuclei can be stored in RNAlater]
QA/QC: Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets.
High clumping rates would indicate damaged nuclei and would require re-filtering using 30-µm CellTrics filter or exclusion from downstream analyses. At least 50,000 nuclei are needed to proceed with snDrop-seqAt least 10,000 nuclei are needed to proceed with 10X 3’ RNA v3
?. [snRNA-Seq methods: nuclei can be stored in RNAlater]
To remove RNAlater, centrifuge nuclei at 4000g,  at . Remove solution and resuspend in associated nuclei resuspension buffer (assay dependent)
4 °C