GSM7822360: WT_rep2; Mus musculus; RNA-Seq
At 3 days following injury, mice were perfused with ice-cold saline solution, and eyes (n=16 WT eyes and n=16 Itgam-/-) were harvested and kept on ice. For two replicates per genotype, 8 eyes were used per sample. Eyes were cut open to collect vitreous humor in a 1.5 ml tube, and in a separate tube, retinas were collected. Retinas were subsequently digested in collagenase (1mg/ml) at 37°C for 30 min and manually triturated with a wide-bore pipette tip to generate a single-cell suspension. To block gene transcription, actinomycin D was added to the digestion solution. Cells from vitreous were pelleted by centrifugation at 500g and combined with retinal cells. To enrich for immune cells, cells were run over an anti-CD45+ magnetic beads column (Miltenyi, 130-052-301). Cells were eluted and counted with a hemocytometer. The Chromium Next GEM Single Cell 3' Reagent kit v3.1 (Dual Index) was used. Barcoding and library preparation was performed following the manufacturer's protocols. Briefly, to generate single-cell gel-bead-in-emulsion (GEMs) solution, approximately 15,000 cells, in a final volume of 43 µl, were loaded on a Next GEM Chip G (10x Genomics) and processed with the 10x Genomics Chromium Controller