GSM4103393: EIU2w-1; Mus musculus; RNA-Seq
Retinal single cell suspensions were re-suspended in 200 µL FACS buffer containing DRAQ7 (DR77524; Biostatus, Shepshed, UK). Live microglial cells (tdTomatohi DRAQ7-) were immediately sorted using a BD Influx Cell Sorter. Retinal samples were prepared in small batches in order to maintain cell viability and ensure high quality RNA.  Cells were sorted into 0.2 mL endonuclease-free tubes containing 0.05 µL RNase inhibitor, 0.95 µL lysis buffer, and a variable amount of nuclease-free water depending on the number of cells collected: 9.5-(x/850*3) µL, where x is the number of cells isolated, using components of the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA). Samples were sorted in a constrained randomised order in blocks; blocks were made as small as possible and consisted of a balance of every experimental group to ensure that time- or order-dependent (batch) effects of sorting were mitigated.   cDNA generated using SMART Seq v4 Ultra Low Input RNA Kit. cDNA isolated using Agencourt AMPure XP DNA beads. cDNA quantified by Agilent Bioanalyser. Nextera XT DNA Kit -- as per recommendations, used 150 pg as input amount.