GSM7468636: Retina, WT, 10X V3 Chemistry; Danio rerio; RNA-Seq
3 fish of the same strain were collected in the morning between nine and eleven a.m., 2 to 4 hours after light onset in the fish room, in Falcon tubes containing 0.15% Tricaine/MS222 and incubated on ice for 10 minutes. Zebrafish were decapitated and eyeballs were detached and placed in dissecting medium containing Leibovitz's L-15 medium (Gibco 21083027). Each retina was then extracted and placed in a 1.5 mL microcentrifuge tube with 300 µL of dissociation solution containing L15 and Earle's Balanced Salt Solution (EBSS; Gibco 14155063) in a 3:1 ratio. After all retinas were collected, the dissociation solution was replaced with 200 µL of Papain solution (50 U/mL in EBSS; Worthington Biochemical LK003178) and retinas were incubated at 28˚C. After 20 minutes of incubation, retinas were gently triturated 20-30 times with fire-polished glass pipettes. Supernatants were collected and incubated with Papain for another 40 minutes with trituration performed every 10 minutes. Papain reaction was terminated by adding 2x volume of 0.1% BSA in L15: EBSS medium. Tubes were centrifuged at 400g for 5 minutes to pellet the cells. The supernatant was removed and washed again with 0.05% BSA. The supernatant was removed and cells were finally suspended in 100 µl of 0.05% BSA. The cell samples were submitted for 3' sc-RNA library preparation and sequencing through the Baylor College of Medicine Single Cell Genomics Core facility. Single-cell Gene Expression Libraries were prepared usingChromium Next GEM Single Cell 3' Reagent Kits v2 and v3.1(10x Genomics, Pleasanton, CA). In brief, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions) where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMS are broken and cDNA from every single cell is pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing and adaptor ligation. The final library was generated by amplification. cDNA libraries were sequenced on an Illumina HiSeq2500. 3' SC RNA Seq