GSM9291241: P20 cortical interneurons in control and in test condition with TeNT in Fezf2+ PNs; Mus musculus; RNA-Seq
Brain samples were harvested from mice with specific genotypes and ages, and the cortex was carefully dissected in ice-cold Homogenization Buffer (HB) comprising 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, 1 mM DTT, 0.15 mM spermine, and 0.5 mM spermidine. The dissected brain tissue was then transferred to a 2 ml Dounce tissue grinder filled with HB supplemented with 0.15-0.25% IGEPAL-CA630 and 0.2 U/µl RNasin. Tissue homogenization was performed with 8-10 strokes of pestle A followed by 9-10 strokes of pestle B. The homogenate was filtered through a 30 μm filter into a 15 ml conical tube and centrifuged at 500 g for 5 min. at 4 °C using a swinging-bucket centrifuge. The resulting pellet was resuspended in 1X PBS containing 1% BSA + 0.2 U/µl RNasin and then passed through a 40 μm filter. DRAQ5 (BioLegend) was added to the nuclei suspension for sorting of GFP+ nuclei on a Sony MA900 cell sorter using a 70 µm nozzle. Nuclei were collected in pre-chilled 0.2 ml PCR tube and counted using a hemocytometer (INCYTO C-Chip). snRNA-seq libraries were prepared using the Chromium Single Cell 3' Kit v3.1 (10x Genomics), following the manufacturer's protocol. Pooled libraries were sequenced on NovaSeq 6000 instruments (Illumina).