GSM8147318: AVC, IWR1, scRNAseq; Danio rerio; RNA-Seq
Hearts were resected from 1-month-old Tg(fli1:EGFP);Tg(myl7:GAL4db-TCFΔC);(5xUAS:NTR-mCherry) fish treated with DMSO or 10 μM MTZ for 48 h. The resected hearts were collected in 35-mm dishes containing HBSS without calcium and magnesium. The atrium and the ventricle were removed from AVCs. Only AVCs were transferred to a 12-well plate. The AVCs were digested with 3 mg/ml collagenase type IV (Worthington, LS004188)/HBSS for 30 min at 28.5 ºC. After pipetting, the cells were collected in low-binding tubes, pelleted (3000 rpm, 5 min at 4ºC), and washed in 0.5% FBS/DMEM (phenol red free). Cells were resuspended in 0.5% FBS/DMEM and counted using a cell counter (Thermofisher Scientific). The resulting cell suspension was loaded onto a 10X Chromium Controller (10X Genomics). The scRNA-seq library was prepared using Chromium Next GEM single cell 3'reagent kits v3.1 (10X Genomics, 1000128), according to the manufacturer's protocol. Following droplet generation and barcoding, cDNA was synthesized and amplified. The cDNA was processed to construct Illumine sequencing libraries. Sequencing was performed on a NextSeq 500 (Illumina). Raw sequencing data were processed using 10X Genomics Cell Ranger software (v6.1.1).