GSM6625507: Infected, scRNAseq; Danio rerio; RNA-Seq
Euthanized adult zebrafish were briefly dunked in room temperature phosphate-buffered saline (PBS, Gibco 14190-144) and then placed in a 60mm Petri Dish (Fisher FB0875713A) containing 8 milliliters of room temperature PBS. One (gloved) hand was used to hold the fish in place by firmly grasping the head, and the second hand was used to scrape rostrally along the body of the fish with an angled dissecting knife (Fine Science Tools 10056-12) to descale. This was done under a dissecting microscope and scale loss was visually monitored. Scales at the base of the caudal, dorsal, and anal fins typically had to be individually removed by plucking with thin-tip tweezers (Dumont). PBS from the Petri Dish was then washed over the scaled fish for approximately 30 seconds with a transfer pipette (Fisher 13-711-7M) to promote cells entering the suspension, and the buffer was then passed through a 40-micron filter. This cell suspension was then mixed 1:1 with cell isolation media (L-15, penicillin-streptomycin, and 2% FBS) in a 15 mL falcon tube, washed, resuspended in 500 uL of ACK lysis buffer for 90 seconds to lyse red blood cells, and washed once more with cell isolation media. Library was performed according to the manufacter's instructions (Chromium Single Cell Gene Expression Solution 3' v2, 10x Genomics).