GSM7986397: LXB4; Mus musculus; RNA-Seq
Retina was dissociated to single-cell suspension using a papain dissociation kit (Worthington, Columbus, OH). Briefly, dissected retinas were placed in papain solution for 30 mins at 37 oC with exposure to 5% CO2 in the incubator. Retinas were gently tapped and incubated for 15 more mins for complete dissociation. The reaction was quenched by adding ovomucoid inhibitor solution, and cells were collected after centrifugation and dissolved in resuspension buffer containing PBS+5%BSA+ DNASE to form a single-cell solution of cells. Cells were passed from a 40mM cell strainer (Sigma) to remove debris and clumps. Before processing the cells for single-cell transcriptomics, rod cells were depleted to enrich the rest of the population. Cells were incubated with Biotin-CD133 antibody for rod-specific labeling and were depleted in the magnetic column after anti-biotin-magnetic bead labeling (Miltenyi Biotech, Bergisch Gladbach, Germany). Barcoding and library preparation were done using a 10X Chromium Single Cell 3' reagent (v3.1 chemistry, 10x Genomics, Pleasanton, CA) per the manufacturer's instructions. Briefly, Cells were loaded on the chromium chip to create gel beads containing unique oligo barcodes with single cells, creating cell-gel droplets. RNA from each cell was captured within the droplets, and cell-specific barcodes were linked to their respective transcripts. Reverse transcription and amplification of captured RNA were performed to generate cDNA libraries. Quality of cDNA preparation was checked using Bioanalyzer (Applied Bioscience)