GSM4090373: RGC061616p1_E11_S59; Mus musculus; RNA-Seq
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)
GEO Accession
GSM4090373