GSM6213612: Srf-Ctrl, replicate 3; Mus musculus; RNA-Seq
Eye balls from P12 pups were harvested and collected in ice cold MEM (Gibco, #31095-029) supplemented with 25 mM HEPES (Thermofisher, #15630049), penicillin/streptomycin (Gibco, #15140) and 10% fetal calf serum (FCS). Retinas were subsequently dissected and enzymatically digested using the Papain Dissociation Kit (Worthington Biochemical Corporation, #LK003150). After digestion, the single-cell suspension was filtered through a 40 µm cell strainer into a 2 ml reaction tube. Hereafter, the filtered cell suspension was centrifuged for 5 min at 300 x g at 4 °C and the supernatant was carefully removed. The remaining cell pellet was resuspended in 120 µl of FACS buffer (2% FCS and 2mM EDTA pH 8.0). 100 µl of the resuspended cells were mixed with 100 µl of FACS buffer containing fluorescently labeled ABs (Ter119-PB, CD45-PB, CD140a-FITC, CD140b-APC and CD31-PE, final dilutions see Online Table I), followed by incubation for 30 min on ice. The remaining 20 µl of whole retina single-cell suspension was kept as an input control and lysed in 350 µl of RLT buffer plus (Quiagen) supplemented with β-mercaptoethanol (10 µl/ml).  After AB incubation, 1 ml of FACS buffer was added to the cell suspension to wash out unbound ABs, followed by centrifugation for 5 min at 300 x g at 4 °C. Finally, the supernatant was carefully removed and the remaining cell pellet was resuspended in 500 µl FACS buffer containing 50 ng/ml DAPI (Sigma, #D9542) to label dead cells. Labeled single cell suspensions were sorted subsequently on a BD FACS Aria lllu sorter (BD Bioscience) using a 100 µm nozzle. Gating strategies for discrimination of cell populations were based on their immunolabelling defined according to reference experiments using single-stained samples and fluorescence minus one (FMO) controls. 160-590 pg of RNA were used for sequencing library preparation with the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England BioLabs). Libraries were validated in the 2100 BioAnalyzer and quantified by qPCR and fluorometric analysis (Qubit 2.0, Invitrogen).