title: GSM8577299: fetal SAN, snRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: SAN tissue was dissected from the heart. The tissue was immediately frozen using liquid nitrogen and stored at -80°C. The frozen tissue was retrieved and dissociated using lysis buffer containing: 0.32M sucrose (Sigma, S0389), 5mM CaCl2 (Fluka, 21114), 3mM Mg(AC)2 (Sigma, 228648), 20mM Tris-HCl (ThermoFisher, 15567-027), 0.1%Triton X-100 (MilliporeSigma, X100), 0.1mM EDTA (ThermoFisher, AM9260G) in DNase/RNase free water (Qiagen, 129115). A douncer was used to mechanically homogenize the tissue until single nuclei were obtained. Homogenate was washed twice with PBS containing 1% BSA and 0.2U/ul RNAseOUT (ThermoFisher, 10777019) and filtered through a 40um cell strainer. Nuclei were stained with DAPI (10g/ml) and sorted for DAPI+ single-nuclei on a FACSAria (BD) sorter at the SickKids-UHN Flow Cytometer Facility. Library construction was performed according to manufacturers instruction (single cell 3' v3 protocol, 10x Genomics)