GSM7027207: ipRGC, P1, rep192; Mus musculus; RNA-Seq
Retinas were dissected in cold Hibernate A medium lacking Ca2+, Mg2+, and phenol red (BrainBits). Retinas were then dissociated in papain (Worthington Biochemical, LS003126), 5.5 mM Cysteine-HCl, and 1.1 mM EDTA dissolved in Hibernate A medium (lacking Ca2+, Mg2+, and phenol red) for seven minutes at 37°C. Cell suspension was passed through a 40 μm cell strainer (Falcon, 352340) and incubated for 30 minutes on ice with Brilliant Violet (BV)-421 anti-mouse CD90.2 antibody (BioLegend, 105341, 1:300), which binds to RGCs and provides an additional selection marker during FACS. The cells were then centrifuged and resuspended in 0.4% bovine serum albumin dissolved in Hibernate A medium (lacking Ca2+, Mg2+, and phenol red). Cells were again centrifuged and resuspended in 2000 U/ml DNase I (New England Biolabs, M0303S) in Hibernate A medium (lacking Ca2+, Mg2+, and phenol red) prior to cell sorting. Individual GFP+, BV-421+ cells were sorted into a lysis solution consisting of 3500 U/ml RNase inhibitor (New England Biolabs, M0314S), 140 U/ml DNase I, and 0.17% v/v Triton X-100 in water using a MoFlo Legacy cell sorter (Beckman Coulter). Single-cell DNA libraries were prepared according to the Smart-seq2 protocol (Picelli et al., Nature Protocols, 2014) and sequenced using an Illumina NextSeq 500 sequencer (75 bp paired-end reads, 400 million total reads).