GSM7978132: retina, scRNA-seq; Danio rerio; RNA-Seq
Fifty heads were dissected from 4-dpf heterozygous Tg(crx:GFP)stl887Tg larvae. Following dissection, eyes were stored in ice-cold Hanks' Balanced Salt Solution (HBSS) until all eyes were harvested. Once the eyes were collected, HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium-free HBSS containing 0.4 mg papain (Worthington Biochem) for 15 min at 37°C. 800 µl of 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) containing 5mM MgCl2 and 120 units DNaseI (Roche) were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer (2.5 mM EDTA, 25 mM HEPES, 1% bovine serum albumin (BSA) in calcium/magnesium-free HBSS). Cells were sorted on an Aria II FACS machine (BD biosciences) with gating based on forward scatter, side scatter, and GFP fluorescence and collected in 700 μl of D-PBS without Ca2+ and Mg2+  supplemented with 0.4 % BSA (D-PBS-CMF) in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min, washed with D-PBS-CMF, centrifuged, and supernatant reduced to ~80 µl. Cell density was quantified on a hemocytometer, and ~ 5000 cells were used for single-cell library preparation. A library for single-cell RNA-seq was constructed with the Chromium v3  platform  (10X  Genomics,  Pleasanton,  CA) according to the manufacturer protocol.