GSM9555819: AAV-Yap5SA Sample 3; Mus musculus; RNA-Seq
Retinas were dissected in ice-cold PBS and enzymatically dissociated using 10 units of papain and 60 units of DNase (500 μL/30 μL for 3-6 retinas) for 25-30 minutes in a shaking water bath at 37°C. Following digestion, tissues were gently triturated 10 times with a fire-polished pipette, and the reaction was quenched by adding EBSS containing ovomucoid and DNase. The resulting cell suspension was filtered through a 40 μm strainer and centrifuged at 900 RPM for 8- 10 minutes. For fluorescent-activated cell sorting (FACS) prior to single-cell mRNA sequencing, cell pellets were resuspended in a solution of DNase, ovomucoid, and Neurobasal medium (1:1:10) and kept on ice. FACS was performed using a Sony MA900 cell sorter (BD Biosciences) with an 80-micron nozzle to isolate tdTomato-positive and -negative cells. Sorted cells were collected into pre-chilled tubes containing 0.04% ultrapure BSA in 0.1 M PBS and immediately processed for single-cell RNA sequencing; Library construction was performed according to the manufacturer's protocol (10x Chromium Single Cell 3' v3.1). Single-cell RNA sequencing was performed using the 10X Genomics Chromium platform. Cell concentration and viability were assessed, and approximately 10,000 viable cells per condition were loaded onto the Chromium Controller for single-cell capture and barcoding. Following reverse transcription and barcoded cDNA amplification according to the manufacturer's protocol, sequencing libraries were prepared and submitted to Admera Health (South Plainfield, NJ) for sequencing. Libraries were pooled and sequenced on an Illumina NovaSeq X Plus platform using 10B flow cells with 2×150 bp paired-end sequencing (NovaSeq X Plus 10B 2×150 configuration) to achieve a target depth of approximately 50,000-100,000 reads per cell. Demultiplexing was performed by the sequencing facility, and raw sequencing data were delivered via Illumina BaseSpace with MD5 checksums and quality control summary reports.