title: GSM9065633: FACS sorted epicardial cells (CD31-tdTomato+PDPN+), Biological replicate sample 1, E11.5; Mus musculus; ATAC-seq
library_name: GSM9065633
library_construction_protocol: Whole embryonic hearts were enzymatically dissociated using the Neonatal Heart Dissociation Kit (Miltenyi Biotec) following the manufacturer's instructions and as previously described. Biological replicate samples are indipendent samples generated from pooled hearts.E11.5, E13.5, and E17.5  hearts (Rosa26tdTomato; Wt1CreERT2/+) from littermate embryos were pooled prior to dissociation (E11.5: 6 hearts/sample; E13.5: 4-13  Hearts/sample; E17.5: 4 Hearts/sample) to maximise cell numbers for FACS and corresponding FMO controls, and achieve a target sort of 50,000 epicardial cells/sample for the downstream ATAC-seq protocol. Single-cell suspensions in cell staining buffer (Biolegend) were pre-treated for 5 minutes on ice with TruStain FcX antibody, followed by surface staining with BV421 anti-CD31 and APC/Cy7 anti-Podoplanin (PDPN) antibody for 30 minutes on ice, then washed with PBS before being incubated with SYTOX Green Dead Cell Stain for 5 minutes on ice. After PBS washes, cells were resuspended in sorting buffer containing 2% FBS in PBS. BD FACSAria III was used to sort 40,000-50,000 live cells negative for SYTOX Green and CD31, and positive for tdTomato and PDPN, into 0.5% BSA in PBS. Live epicardial cells (CD31-tdTomato+PDPN+) from E11.5 and E17.5 hearts and live epicardiium-derived cells (EPDCs) (CD31-tdTomato+PDPN-) from E12.5 and E13.5 hearts were isolated by FACS were subjected to the ATAC-seq protocol as previously described. ATAC-seq DNA libraries were generated as previously described, with minor modifications. Reactions were purified using the MinElute Reaction Cleanup Kit (Qiagen) following the manufacturer's instructions. Pre-amplification of transposed fragments (5 cycles) was performed using NEBNext Ultra II Q5 Master Mix (NEB) and Nextera i7 index adapters (IDT). The number of additional amplification cycles (typically 4) was determined using the qPCR method. Final PCR products were purified using a double-sided bead purification method with SPRIselect beads (0.5X volume followed by 1.3X volume of SPRI beads). Final cleanup was performed with the MinElute Reaction Cleanup Kit (Qiagen). Libraries were assessed for quality using an Agilent TapeStation with a D1000 DNA ScreenTape assay, and quantified with the KAPA Library Quantification Kit. Next-generation sequencing (NGS) was conducted on equimolar pooled libraries using the NextSeq 500/550 High Output v2.5 kit (75 cycles) (Illumina), with paired-end reads (40 bp x 2) and 8 bp single index reads.
identifiers.namespace: GEO
identifiers.id: GSM9065633_r1