Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Retinas were dissected in oxygenated AMES, digested in papain, and dissociated to single cell suspensions using manual trituration in ovomucoid solution. Single cell suspensions were incubated with 0.5ul of anti-CD90 (conjugated to various fluorophores) (Thermo Fisher Scientific) for 10 million cells per 100µl. Cells were sorted using a MoFlo Astrios. Cellular debris, doublets, and dead cells were excluded, and RGCs were collected based on high Cd90 and GFP co-expression. Singe cells were sorted into 2ul lysis buffer per well in multiple 96 well plates, spun down for 1min and immediated placed on dry ice, then stored at -80oC until subsequent processing for Smartseq2 We generated single cell RNA-Seq libraries using a modified Smart-seq2 method [Picelli et al., 2013, Ding et al., 2019] with the following minor change. We added 3 µl instead of 4 µl of master mix containing only 1.7 µl instead of 2.7 µl of 1 M Trehalose (Sigma-Aldrich) directly to the 2 µl cell lysate without a SPRI bead cleanup step. Smart-seq2 per Picelli et al., 2013 with minor modifications (see above)