title: GSM6205478: BKSWT, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Both BKSDB (n = 2) and BKSWT (n = 2) mice were euthanized with cervical dislocation at 13 weeks of age. Tape the limbs of the mouse in supine position, spray the skin of the head area with 75% ethanol. Retinas of two eyes were isolated carefully and enzymatically digested to generate single-cell suspensions for scRNA-seq. The concentration of single cell suspension was determined using the Count Star instrument and adjusted to 1000 cells/ul. Using single cell 3' Library and Gel Bead Kit v3.1 (10x Genomics, Cat. No. 1000121) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, Cat. No. 1000120), the cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. In brief, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cell will be recovered (about 3000 cells). Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed in 200 ul tube (NEST Biotechnology, Cat. No. 401001) on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 minutes, followed by 85°C for 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using the Agilent 4200. The cDNA libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with paired-end 150 bp (PE150) reading strategy.