TITLE: Nuclei Isolation for 10x Chromium single-nuclei RNA sequencing
AUTHORS: Chiara Pavan, Clare Parish

[DESCRIPTION]
Single-nucleus RNA sequencing (sn-RNA seq) enables the profiling of nuclear gene expression in isolated cells. Herein, we present a step-wise protocol for single nuclei isolation from a fresh-frozen small biopsy of rat brain containing a human xenograft. The described method includes human-neuronal nuclei isolation and debris removal using fluorescent-activated
nuclei sorting. The isolated nuclei were processed through the 10x Chromium Controller platform for snRNA-seq. Compared to single-cell RNA-seq (sc-RNA seq), the use of nuclei avoids dissociation-associated transcriptional artefacts and is
compatible with frozen tissue.




[GUIDELINES]
General notes

Input nuclei suspension should contain >90% dead cells with intact nuclei. The presence of a high fraction of dying cells with damaged nuclei per ambient RNA and cellular debris may influence sequencing performance


Lysis
There are various types of lysis buffer that can be used to lyse the cells and release the nuclei. This very much depends on the tissue type. The basic buffer can either be hypotonic (which might help lyse the cells and release the nuclei) or isotonic (which may be more intrinsically gentle for the preparation). The detergent that is used to lyse the cells can be varied (as well as the concentration). Commonly used buffers alternative to the one we used are:

Lysis buffer NST (isotonic+NP40)
10mM Tris HCl pH 7.4
146mM NaCl
3mM MgCl2
0.1% NP40
40U/ml of RNase inhibitor

Lysis buffer IST (hypotonic + NP40)
 10mM Tris HCl pH 7.4
10mM NaCl
3mM MgCl2
0.1% NP40
40U/ml of RNase inhibitor

For every type of tissue, the number of strokes with the dounce homogenizer needs to be calibrated. The incubation time after the dounce homogenization can also be varied. For each tissue an optimization needs to be performed.


Tubes
Nuclei as opposed to cells are problematic since they are smaller and they are more sticky because of DNA leakage and other factors. This means that there is much more significant loss after each centrifugation step. To try to minimize the loss, we have tried various methods to prevent the nuclei sticking to the sides of the tubes. One that work and we recommend is the coating of the tubes for about one hour before use with PBS+1%BSA. Recently, we have tried using polystyrene 5ml FACS tubes but we cannot comment if they are any better at reducing loss.


[STEPS]
SECTION: Preparation of reagents and materials
1. Prepare ~50 mLof ice-cold PBS and store it on ice
Prepare 8 mLof ice-cold lysis buffer containing 0.2U/ul of RNase inhibitor by adding 40 µL of RNase inhibitor to 8 mLof Nuclei EZ-prep
Prepare 10 mLof ice-cold wash buffer containing 1%BSA and 0.2U/ul of RNase inhibitor by adding 2 mLof 5%BSA and 50 µL of RNase inhibitor to 8 mL of 1xPBS
SECTION: Transcardial perfusion
2. Perfuse the animal with ice-cold PBS
Rapidly and on ice, extract the brain and using fine forceps, dissect the rat striatum containing the human xenograft 
Immediately transfer the tissue in an ice-cold 1.5 mL tube and snap-freeze in in liquid nitrogen or dry ice
Store the sample to -80 °Cuntil use


SECTION: Nuclei isolation
3. on ice Transfer the tissue from the -80 °C to the dounce homogenizer containing 1.5 mL of cold lysis buffer on ice
Homogenize with 24 strokes on ice
Transfer the homogenized sample to a 15ml Corning tube on ice
Wash the dounce with 1.5 mLof lysis buffer
Allow the tube to stand for 5 min onon ice
Centrifuge at 500 x g for 5 min at 4 °C
Remove the supernatant
Wash with 1 mLof lysis buffer on ice
Wait 5 min on on ice
Centrifuge at 500 x g for 5 min at 4 °C
Remove the supernatant
Resuspend in 1 mL wash buffer (gently, without creating bubbles) on ice
Filter with a 30um cell strainer (MACS SmartStrainers)on ice
Wash the strainer with 1 mL wash buffer on ice
Centrifuge at 500 x g for 5 min at 4 °C
Remove the supernatant
Resuspend in300 µL wash buffer on ice
Count the nuclei. We recommend using both Trypan Blue to assess nuclei integrity and a fluorescent cell counter When looking at the nuclei in the counter, look at the integrity of them – how round they are with clear border and how much they are single and dispersed and not in clumps or in small groups of cells
Stain using HNA-PE and NeuN-Rb (1:100 diluted in wash buffer)
Incubate for 30 min at 4 °C in the dark
Add secondary antibody (Rb-647, 1:200)
Incubate for 20 min at 4 °C in the dark
Wash with 1 mL of wash buffer on ice
Centrifuge at 500 x g for 5 min at 4 °C
Wash with 1ml of wash buffer 20 min
Centrifuge at 500 x g for5 min at 4 °C
Resuspend in DAPI solution (dilute the stock of 1mg/ml in 1:1000, 1ul in 1ml)
FACS sort the nuclei in 35 µL of wash buffer using a 70 μm nozzle, 21– 22 p.s.i. The sort should be done for HNA+ NeuN+ DAPI+ single nuclei
After FACS sorting, centrifuge the nuclei 600 x g for 8 min
Carefully remove as much supernatant as possible and count in a cell counter. Again, look at the integrity of the nuclei. The nuclei should be counted twice and the average concentration calculated.
The nuclei are now ready for a 10x run. In our case sample processing was performed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomic, California, # PN-1000121) and
the Chromium Controller (10X Genomics, California) per manufacturer's instructions as published in User Guide CG000204 Rev D (10X Genomics,California)