GSM7109897: OIR Socs3 cKO P13, scRNAseq; Mus musculus; RNA-Seq
The fresh retinas were isolated from mouse eyes and the single cells were generated by Papain Dissociation System (Worthington-biochem LK003150). Retina myeloid cells were incubated with FITC-CD45 antibody for 30 min on ice in the dark box. Wash the cells 3 times by centrifugation at 1000 rpm for 3 minutes and resuspend cells in ice-cold FACS buffer for sorting. Dead cells were removed during sorting. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.