GSM8997253: WT zebrafish neurons; Danio rerio; RNA-Seq
Twenty-two old TgBAC(neurod1:EGFP)nl1 zebrafish embryos were collected and euthanized in 1.7 ml microcentrifuge tubes. Embryos were deyolked using a calcium-free Ringer's solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES pH 7.0), by gently pipetting up and down with a P200 pipet. Embryos were incubated for 5 minutes in Ringer's solution. Embryos were transferred to pre-warmed protease solutions (0.25% trypsin, 1 mM EDTA, pH 8.0, PBS) and collagenase P/HBSS (100 mg/mL) was added. Embryos were incubated at 28° C for 15 minutes and were homogenized every 5 minutes using a P1000 pipet. The Stop solution (6X, 30% calf serum, 6 mM CaCl2, PBS) was added and samples were centrifuged (350xg, 4° C for 5 minutes). Supernatant was removed and 1 mL of chilled suspension solution was added (1% FBS, 0.8 mM CaCl2, 50 U/mL penicillin, 0.05 mg/mL streptomycin, DMEM). Samples were centrifuged again (350g, 4° C for 5 minutes) and supernatant was removed. 700 μl of chilled suspension solution was added and cells were resuspended by pipetting. Cells were passed through a 40 μm cell strainer into a FACs tube and kept on ice. GFP and RFP+ cells were FAC sorted on a BD Symphony cell sorter into sorting buffer (50 μl PBS/ 2% BSA) in a siliconized 1.5mL tube. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics).