GSM9463399: Week 8 and Week 16 hearts; Nothobranchius furzeri; RNA-Seq
Week 8 and Week 16 Killifish hearts were removed from the body and dissected further to remove atrium and outflow tract while leaving ventricle. Dissected ventricles were quickly transferred to a RNAse-free tube containing tissue dissociation solution (1mL HBSS + 7.5 mg Collagenase/Dispase + 7.5 mg Collagenase type II). Within the tube, dissected ventricles were minced with a plastic pestle for 2 minutes. The tubes were then placed on a rotator at room temperature for 45 minutes and centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed, and the pellet was washed with 400 uL HBSS/10% FBS followed by centrifugation at 1,000 rpm for 2 minutes. The supernatant was removed again, and the pellet was mixed with 400 uL HBSS/10% FBS. Cells were filtered through a 70 μm sterile cell strainer to enrich the population of rarely detected cell types, such as immune cells and epicardium, and about 250 μL of the filtrate was collected in an Eppendorf Protein LoBind tube to assess cell number, morphology, and viability Cell fixation was completed following the Parse Biosciences Evercode cell fixation kit v3 kit protocol. scRNA-seq library preparation was performed using the Parse Biosciences Evercode Whole Transcriptome (WT) kit. Briefly, fixed single cells were loaded across three rounds of combinatorial barcoding processes to generate eight sublibraries. cDNA synthesis, fragmentation, and end repair, Illumina Truseq adapter ligation, and i5/i7 unique dual indexing were conducted for all sublibraries.