GSM8530849: Control; Mus musculus; RNA-Seq
~ 100 mg of frozen liver were used for nuclei isolation and single nucleus RNA sequencing (snRNAseq) analysis. Nuclei were isolated from one Control and one CRISPR/Donor + siRNA (Repair Drive) mouse sacrificed at 12 weeks post-AAV injection. Liver samples were cut into tiny pieces in 250 µL of buffer HB (0.25 M Sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH, pH 7.8) supplemented with 1x DTT, spermine and spermidine, and 0.2 U/µL of Protector RNase inhibitor (Millipore Sigma) (referred as buffer HB++). Then, samples were centrifuge at 500 g for 5 minutes and resuspended in 500 µL of buffer HB++. Samples were homogenized in a Dounce homogenizer firstly with a loose pestle for 30 strokes, followed by a tight pestle for 10 strokes after adding 250 µL of 1% NP-40 (in buffer HB++). Homogenates were filtered through a pre-wet 40 µm cell strainer (Falcon) by adding 10 mL of nuclei wash buffer (NWB, 1% BSA in PBS supplemented with 0.2 U/µL of Protector RNase inhibitor). Samples were centrifuged at 500 g for 10 minutes at 4°C using a swinging bucket rotor and the pellets were resuspended in 500 µL of NWB buffer and filtered through a 40 µm cell strainer (Flowmi, Millipore Sigma). 900 µL of sucrose cushion buffer (SCB: 9 volumes of Nuclei PURE 2M sucrose cushion solution and 1 volume of Nuclei PURE sucrose cushion solution, Sigma-Aldrich, supplemented with 0.2 U/µL of Protector RNase inhibitor) were added to the resuspended pellet and the obtained mix was layered on top of 500 µL of SCB in a 2 mL Eppendorf tube. Nuclei were centrifuged at 13000 g for 45 minutes at 4°C. The obtained pellet was resuspended in 1 mL of NWB and filtered through a pre-wet 20 µm CellTrics filter (Fisher Scientific). Nuclei were finally centrifuged at 500 g for 5 minutes and resuspended in 200 µL of NWB buffer. Nuclei were stained with DAPI and nuclei purity and integrity were confirmed using a fluorescent microscope. Samples were diluted to obtain 15000 - 18000 nuclei for single cell library construction according to the 10X single cell gene expression workflow (10X Genomics, Chromium Next GEM Single Cell 3' Kit v3.1, 1000269). Quality of the cDNA and the libraries were analyzed using the high sensitivity NGS fragment analysis kit (Agilent). Libraries were sequenced on an Illumina NovaSeq 6000 instrument.