title: GSM8094162: Control, replicate2, snRNAseq; Mus musculus; RNA-Seq
library_name: GSM8094162
library_construction_protocol: Both striata from each mouse were pooled prior to nuclear isolation. Nuclei were isolated using the 10x Genomics Nuclei Isolation Kit with RNase Inhibitor (10x Genomics) with minor modifications. First, flash-frozen tissue was transferred to ice-cold lysis buffer (from the Nuclei Isolation Kit) in a 1mL glass tissue homogenizer (Wheaton). After douncing, the tissue lysate was resuspended and left to incubate for 10min on ice. Additional steps followed the manufacturer's instructions. During the first wash (after debris removal), the nuclear suspension was incubated with rotation for 20min at 4oC with DAPI staining solution (Abcam) at a final concentration of 5µM. Following a spin and final resuspension, nuclei with 2N DNA content were purified by fluorescence-activated nuclear sorting (Sony Cell Sorter) based on DAPI-fluorescence and collected in Wash and Resuspension Buffer (PBS, 0.5% BSA, and 0.2U/µl RNase Inhibitor - prepared according to kit instructions). Sorted 2N nuclei event count and final volume was used to inform sample loading volume, and the nuclei were immediately loaded onto 10x Chromium Single Cell 3' Kit v3.1 chips at ~20k nuclei per sample (this loading concentration was optimized based on sorter nuclear count and 10x nuclear recovery). Libraries were generated following the manufacturer's protocol (single cell 3' v3 protocol, 10x Genomics)