GSM6499490: Retina, 20hr, replicate 2; Mus musculus; RNA-Seq
Isolated retinas were placed in 1-2 mL each of digestion buffer, consisting of Hibernate medium (HBSS; 10-547F, Lonza) supplemented with 5% fetal bovine serum (FBS), 10 mM HEPES, 0.7 mg/mL calcium chloride, 1.5 mg/mL collagenase A (Roche), and 0.1 mg/mL DNase I (Roche). Tissue was incubated at 37 °C for 15-20 minutes, with mild tricheration with a pipette. After digestion, the cell suspension was filtered through a 70 mm cell strainer (Genesee Scientific), and resuspended in PBS. Cells were then stained for viability (Zombie NIR Fixable Viability Kit, 423106, BioLegend), blocked for 5 minutes with Fc block 1 ul/sample (eBioscience) supplemented with 5% each normal rat and mouse sera, and then incubated with Biolegend antibodies raised against CD45 and CD11b diluted in Brilliant Stain Buffer (BD Biosciences) for 15-30 minutes at room temperature. After staining, cell suspensions were washed in 0.5% bovine serum albumin (BSA) with 1:50 EDTA, and ultimately resuspended in the same buffer. Cell sorting was performed at the UC Davis Flow Cytometry Shared Resource Laboratory on an Astrios EQ Cell Sorter (Beckman Coulter). Retinal immune cells were enriched by sorting for alive, CD45+ CD11b+ cells. Cells were sorted into a solution of Dulbecco's Modified Eagle Medium (DMEM; Thermo Fischer) supplemented with 10% FBS, and then immediately used to create the expression libraries.  Library preparation was performed following manufacturer instructions. 10X Genomics (Chromium) single-cell expression 3' v2 chemistry was used. Library preparation was primarily performed at the UC Davis DNA Technologies and Expression Core facility.