GSM9180732: P7 heart; Mus musculus; RNA-Seq
Hearts from P1 and P7 mice were harvested for cell dissociation and live cells FACS sorting. Briefly, for each condition, 3 hearts collected from the same litter were pooled together and finely minced with a scalpel. Tissue was then digested by gentle agitation (180 rpm shaker) in Collagenase II (Gibco,) using a solution of 500 units/ml in HBSS at 37 °C for 45 minutes (P7 hearts) or 20 minutes (P1 hearts). Cell solutions were then passed through a 70 μm filter, washed and incubated in Red Blood Cell lysis buffer (Cell Signaling Technology) for 10 minutes at RT to remove red blood cells. Finally, isolated single cardiac cells were centrifuged, resuspended in 2% FBS in PBS, passed through the filtering cap of the FACS tubes and incubated with 1% 7-AAD viability stain (Invitrogen) for 10 minutes. Approximately 100,000 live cells per sample were sorted using the BD FACSAria Fusion Sorter. Single cell RNA-seq libraries were generated using Single Cell 3 Prime Reagent Kits v1.3 (10x Genomics) according to the manufacturer's protocol. Sequencing was performed on an Illumina NovaSeq 6000 System operated by the Oxford Genomics Centre at the Wellcome Centre for Human Genetics.