GSM8659073: snRNA, Mouse, OIR Model, retina, P17; Mus musculus; RNA-Seq
Retinas from P17 mice under the OIR model were digested using 1 mg/mL Collagenase/Dispase (10269638001; Roche, Basel, Switzerland) and 60 U/mL DNase I (4716728001; Roche) in cell culture medium at 37°C for 15 minutes. The resulting single-cell suspension was filtered through a 40-µm cell strainer and rinsed with PBS containing 2% BSA. The methods for isolating, rinsing, counting, and quality control of the nuclei from cell suspensions adhered to the 10x Genomics user guide for complex tissues (CG000375; 10x Genomics, Pleasanton, CA, USA). In short, the tissues were treated with cold NP-40 lysis buffer in the presence of protease and RNase inhibitors to release nuclei from cells. Purification of the nuclei was carried out using 0.1× lysis buffer, followed by washing with a wash buffer that included RNase inhibitor and 0.1% TWEEN 20 before the permeabilization step. After the diluted nuclei buffer was resuspended, manual hemocytometry was employed to count the nuclei to achieve a targeted nuclei recovery of 10,000 nuclei. The preparation of combined single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) libraries was conducted following the manufacturer's guidelines using the 10x Genomics Single Cell Multiome ATAC + Gene Expression kit.