GSM9139427: Retina, 8w, Rs1KO; Mus musculus; RNA-Seq
Each pooled retina was incubated in 1 mL of digestion solution containing 20 U/mL papain (Worthington Biochemical Corporation, Lakewood, NJ) and 0.005% DNase I (Sigma-Aldrich, St. Louis, MO) at 8 °C for 40 minutes, followed by an additional 10-minute incubation at 28 °C. After enzymatic digestion, the solution was carefully removed without disturbing the tissue. To inactivate enzymatic activity, 700 μL of pre-warmed inactivation solution was added. The tissue was then gently dissociated by pipetting 10 times. Subsequently, 700 μL of washing solution was added, and the mixture was centrifuged at 200 × g for 5 minutes at 4 °C. The supernatant was discarded, and the pellet was resuspended in 3 mL of DPBS containing 0.04% BSA (Gibco, Waltham, MA). The resulting cell suspension was filtered through a 40 μm cell strainer (Corning, Corning, NY) and immediately examined under a brightfield EVOS microscope (Thermo Fisher Scientific, Waltham, MA) to assess cell morphology and viability. The samples were processed for library preparation using Chromium GEM-X Single Cell 3p RNA library v4 (10× Genomics, Pleasanton, CA). After cell dissociation and viability assessment, the cells were mixed with master mix and loaded onto a chromium GEM-X chip with Single Cell 3′ v4 Gel Beads and Partitioning Oil. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets. The cDNA molecules were pooled and processed for library construction. The libraries were then quantified using quantitative PCR and qualified using Agilent Technologies 4200 TapeStation (Santa Clara, CA). Finally, the libraries were sequenced using the HiSeq platform (Illumina, San Diego, CA).