GSM3231460: Input mRNA; Mus musculus; RNA-Seq
Ten-week old wild type C57BL/6J male mouse was purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mouse was sacrificed with cervical dislocation under anesthesia, and an eyeball was immediately removed and placed in cold phosphate buffered saline (PBS). The mouse retina was carefully removed under a dissecting microscope, and the retina tissue was dissociated straightaway using the Papain dissociation system (Worthington, Lakewood, NJ, USA) following the manufacturer's instructions. Briefly, the mouse retina was incubated at 37°C for 30 min in Earle's Balanced Salt Solution (EBSS) with DNase followed by tissue trituration with a 10 ml pipette. Cell pellet was collected after centrifugation at 300g for 5 minutes, and then re-suspended in DNase dilute albumin-inhibitor solution.The cell suspension was carefully layered on top of the albumin-inhibitor solution, and then centrifuged at 70g for 6 minute. Similar procedure was followed for post-morten human eye tissue   Next, the cell pellet was washed and re-suspended in 1 : 1DMEM/F12 + 10% FBS. All centrifugation steps were performed at room temperature. The final cell suspension was filtered with 40 μm cell strainer (Falon, Corning, NY, USA) to remove large clump and debris. All experimental procedures approved by University of Pennsylvania Animal Care and Use Committee. To assess cell viability, cells were stained with 0.4% trypan blue (Mediatech, Inc., Manassas, VA, USA) and counted using a hemocytometer. Retinal Horizontal Cells (RHCs) were identified by means of Lhx1+ and Prox1+ expression. sc-RNA seq was carried out on the viable cells using Chromium 10x Genomics as per the manufacturer's recommendations.
GEO Accession
GSM3231460