title: GSM9393672: Day 1 IFNb Animal 2 Replicate2; Mus musculus; RNA-Seq
library_construction_protocol: Neural Tissue Dissociation Kit-Postnatal Neurons (MACS, Cat. #130-094-802) was used for retinal tissue dissociation.  Retinas (both RPE and neural retina; n=4 for Null-AAV, n=4 for IFN-b-AAV) were dissected out from the anterior segment under normal light in cold D-PBS w/ Ca2+/Mg2+. The retinas were then transferred into pre-warmed buffer containing Buffer Z and Enzyme P, and then incubated for 15 min at 37°C with gentle shaking using a Thermomixer (Eppendorf, Germany). Next, enzyme mix 2 (Buffer Y plus Enzyme A) was added and the retinas were dissociated by gently pipetting up and down using a cropped plastic transfer pipette. The tissue mixture was incubated for 10 min at 37°C on the Thermomixer before adding an additional 7.5 mL Enzyme mix 2. The retinal tissue was further dissociated by trituration and the suspension was filtered through a 70 mm cell strainer and centrifuged for 10 min at 200g (4°C). Dissociated retinal cells were incubated with anti-CD73-PE (MACs, Cat. #130-102-616, 107 cells per tube) followed by anti-PE microBeads (MACs, Cat. #130-048-801, 107 cells per tube) to partially deplete rods. Calcein violet AM (Invitrogen, USA, Cat. #C34858) and eFluor780 (Invitrogen, USA, Cat. #65-0865) were used to enrich live cells. Incubation was done in the dark at 4°C for 20 min. CD73 negative cells were selected via MS Columns (MACs, Cat. #130-042-201) through a Mini MACs Separator (MACs, Milltenyi Biotec, Germany). Viable CD73 negative cells were further enriched by using a FACS sorter (Aria Fusion, BD Biosciences, USA) with gating of Calcein AM positive, eFluor780 negative cell populations. Single, viable cell suspensions were diluted to 1000 cells/mL in PBS with 0.04% BSA for loading into 10x Chromium Single Cell v 3.1 Chips. Following collection, cDNA libraries were prepared following the manufacturer's protocol and sequenced with a NovaSeq 6000 S1 Reagent kit v1.5.