GSM4627214: Adult_Zebrafish_2; Danio rerio; RNA-Seq
Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 (larval retinas) or 45 minutes (adult retinas). To stop the digestion, the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension, cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence-activated cell sorting (FACS) purification. Non-transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within ~45 minutes after FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of ~30,000 reads per cell. 10x Chromium 3' v2 platform (10X Genomics, Pleasanton, CA)
GEO Accession
GSM4627214