GSM4089148: ActR1GLAST; 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 and CD45 (conjugated to various fluorophores) (Thermo Fisher Scientific) for 10 million cells per 100µl. 30µM ActinomycinD (Millipore Sigma) was added to the oxygenated AMES, papain, and ovomucoid solutions. Cells were then resuspended in AMES + 4%BSA + 3µM ActinomycinD prior to FACS sorting.  Cells were FACS 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. Collected cells were loading into 10X Chromium Single Cell A Chips at ~1,000 cells/ul. Single cell libraries were made with Chromium 3' v2 platform (10X Genomics, Pleasanton, CA) following the manufacturer's protocol. Briefly, single cells were partitioned into Gel bead in EMulsion (GEMs) in the GemCode instrument with cell lysis and barcoded reverse transcription of RNA, followed by amplification, shearing and 5' adaptor and sample index attachment. On average, approximately 10,000 single cells were loaded on each channel and approximately 6,000 cells were recovered. Libraries were sequenced on Illumina HiSeq 2500.
GEO Accession
GSM4089148