GSM5351368: retinal cells; Danio rerio; RNA-Seq
Retinas of five-month-old adult zebrafish were were harvested at around zeitgeber time 3 (ZT 3). The dissected retinas were dissociated with a papain dissociation solution, followed by further incubation with 10% FBS in DMEM and DNaseI. After the trituration, the samples were resuspended in sorting buffer (20 mM HEPES and 0.04% bovine serum albumin in Calcium- and magnesium-free HBSS, pH 7.4). Four retinas from two individuals were combined for Tg(rho:EGFP) fish, while six retinas from three individuals were combined for Tg(gnat2:EGFP) fish. The filtered samples were incubated in a solution of propidium iodide and Hoechst 33342. GFP-positive cells were isolated via fluorescence-activated cell sorting (FACS). Dead cells were then removed based on propidium iodide positivity. Intact rods and cones were then selected based on the presence of both blue (Hoechst 33342) and green fluorescence (GFP). About 35,000 viable, intact GFP-positive cells (PI-, GFP+, Hoechst+) and 1,500 GFP-negative cells (PI-, GFP-, Hoechst+) were collected from Tg(gnat2:EGFP) fish, while 10,000 GFP-viable, intact GFP-positive cells and 1,500 GFP-negative cells were collected from Tg(rho:EGFP) fish. These isolated cells were collected into the sorting buffer in one tube. Approximately 6,000 single cells were loaded into a 10X Chromium Single Cell Chip. Single cell libraries were made with Chromium 3' v3 platform following the manufacturer's protocol. RNA libraries were prepared for sequencing using standard Illumina protocols.