Nuclei Isolation for 10x Chromium single-nuclei RNA sequencing
Clare Parish, Chiara Pavan
ASAPCRN
10x-Chromium
single-nuclei RNA sequencing
nuclei isolation
sn-RNA seq
stem cells transplantation
xenografts
Abstract
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.
Steps
Preparation of reagents and materials
Prepare ~50mLof ice-cold PBS and store it on ice1. Prepare 8mLof ice-cold lysis buffer containing 0.2U/ul of RNase inhibitor by adding 40µL of RNase inhibitor to 8mLof Nuclei EZ-prep
- Prepare
10mLof ice-cold wash buffer containing 1%BSA and 0.2U/ul of RNase inhibitor by adding2mLof 5%BSA and50µLof RNase inhibitor to8mLof 1xPBS
Transcardial perfusion
Perfuse the animal with ice-cold PBS1. 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
1.5mLtube and snap-freeze in in liquid nitrogen or dry ice - Store the sample to
-80°Cuntil use
Nuclei isolation
On ice Transfer the tissue from the -80°C to the dounce homogenizer containing 1.5mL of cold lysis buffer On ice1. Homogenize with 24 strokes On ice
- Transfer the homogenized sample to a 15ml Corning tube on ice
- Wash the dounce with
1.5mLof lysis buffer - Allow the tube to stand for
0h 5m 0sonOn ice - Centrifuge at
500x g,0h 0m 0sfor0h 5m 0sat4°C - Remove the supernatant
- Wash with
1mLof lysis buffer on ice - Wait
0h 5m 0sonOn ice - Centrifuge at
500x g,0h 0m 0sfor0h 5m 0sat4°C - Remove the supernatant
- Resuspend in
1mLwash buffer (gently, without creating bubbles)On ice - Filter with a 30um cell strainer (MACS SmartStrainers)
On ice - Wash the strainer with
1mLwash bufferOn ice - Centrifuge at
500x g,0h 0m 0sfor0h 5m 0sat4°C - Remove the supernatant
- Resuspend in
300µLwash bufferOn 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
0h 30m 0sat4°Cin the dark - Add secondary antibody (Rb-647, 1:200)
- Incubate for
0h 20m 0sat4°Cin the dark - Wash with
1mLof wash bufferOn ice - Centrifuge at
500x g,0h 0m 0sfor0h 5m 0sat4°C - Wash with 1ml of wash buffer
0h 20m 0s - Centrifuge at
500x g,0h 0m 0sfor0h 5m 0sat4°C - Resuspend in DAPI solution (dilute the stock of 1mg/ml in 1:1000, 1ul in 1ml)
- FACS sort the nuclei in
35µLof 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
600x g,0h 0m 0sfor0h 8m 0s - 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)
