GSM3332275
GSM3332275: M2Fovea8; Macaca fascicularis; RNA-Seq
0.5-1.5 mm diameter foveal tissues centering on the foveal pit were dissected out from four macaque eyes. Foveal tissues were digested with 200 units papain (Worthington, LS003126). Foveas M1 and M2 foveas were digested at 37 °C; M3 and M4 foveas were digested at 22 °C). Following digestion, retinas were and triturated into single cell suspensions with 0.04% bovine serum albumin (BSA) in Ames solution. Peripheral retinal pieces were dissected and pooled from all quadrants of the retina. Single cell suspensions were dissociated as described for fovea. Dissociated cells were stained incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102) to deplete rods. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Single cell suspensions were diluted at a concentration of 500-1800 cells/mL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell A Chips.  Human cells were dissociated and treated with anti-CD73 as above. 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. Chromium 3' v2 platform (10X Genomics, Pleasanton, CA)
GEO
GSM3332275
GEO Accession
GSM3332275