GSM6466536: DSGC, P12, Spig1-GFP, up, rep16; Mus musculus; RNA-Seq
Retinas were dissected in cold Hibernate A medium (BrainBits) lacking Ca2+, Mg2+, and phenol red. Retinas were dissociated in papain (Worthington, LS003126), 1.1 mM EDTA, and 5.5 mM Cysteine-HCl dissolved in Hibernate A (no Ca2+, Mg2+, or phenol red) for 30 min at 37°C. Cells were then incubated with Brilliant Violet 421-conjugated Anti-CD11b antibody (1:300; BioLegend, Catalog No. 101251) for 30 min at 4 C to label CD11b+ microglia, which can be CTB-Alexa 647+. Cells were incubated with propidium iodide to label dead cells. Single GFP+ CTB-Alexa 647+ CD11b– and GFP– CTB-Alexa 647+ CD11b– cells were sorted into individual tubes containing 3500 U/ml RNase inhibitor (New England Biolabs), 140 U/ml DNase (New England Biolabs), and 0.17% v/v Triton X-100 in water using a MoFlo Legacy cell sorter (Beckman Coulter, Miami, FL). 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).