GSM8898315: WT, uninjured heart ventricles; Danio rerio; RNA-Seq
Pools of four uninjured ventricles and cryoinjured ventricles at 4 dpci were extracted from WT and fbln2PL-/- zebrafish. Anesthetized fish were dissected and ventricles extracted in calcium-, magnesium-free HBSS (Sigma, Cat. no. H9394) supplemented with 20 U/ml of heparin. Ventricles were washed in fresh HBSS and transferred into 1 ml of 1 mg/ml of collagenase (Gibco, Cat. no. 17101015) dissolved in HBSS for 30 min digestion on a thermal mixer at 32 ⁰C and 700 rpm. Ventricles were mechanically disrupted by pipetting every 10 min. Samples were centrifuged at 2000 rpm for 5 min at RT and supernatants discarded. Cells were digested with 1 ml of 1x TrypLE Express Enzyme (Gibco, Cat. no. 12605010) for 15 min on a thermal mixer at 32 ⁰C and 700 rpm and pipetted every 5 min. Enzymatic digestion was stopped by adding 1 ml of 20% fetal bovine serum in HBSS to extracts. Tissue extracts were strained through a 70 μm cell strainer and centrifuged at 2000 rpm for 5 min at RT. The supernatants were discarded, and pellets were washed with 1 ml of 20% fetal bovine serum in HBSS. After centrifuge at 2000 rpm for 5 min at RT, cells were resuspended in 80 μl of 20% fetal bovine serum in HBSS and strained through a 40 μm cell strainer. Cells were counted and viability (>85%) was assessed using 0.4% Trypan Blue. Cells were kept on ice and immediately processed for scRNA-seq. For scRNA-seq experiment, 14000 cells were used for each sample and the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics, Cat. no. PN-1000269) was used following manufacturer's protocol. Each sample was indexed individually using the Dual Index Kit TT Set A (10x Genomics, Cat. no. PN-1000215).