title: Hi-C Heart EC mus musculus: adult male - replicate 2 - deep
design: Heart tissue was dissected and incubated in enzyme solution containing 4 mg collagenase B (Roche), 2.2 mg hyaluronidase (Sigma), and 10 nM CaCl2 in HBSS (Gibco) for 10 min. Dissociation of the remaining tissue was obtained by repetitive pipetting. The resulting suspension was filtered using a 100 m cell strainer and centrifuged at 1000 x g for 5 min at 4 C. Cell pellets were resuspended in PBS containing 2 M EDTA. Cell suspensions were labelled using the far-red fluorescent dye Draq5 (Cell Signaling Technology). Endothelial cells were stained using fluorescein-conjugated Griffonia simplicifolia lectin (Vector Labs). Sorting was carried out using a BioRad S3 Cell Sorter. Cardiac endothelial cells were identified by Draq5 positivity, forward light scatter, and fluorescein intensity. 400,000 to 500,000 endothelial cells per biological replicate were sorted into PBS, centrifuged (1000  g, 5 min), and the pellet snap frozen. Cell pellets were used for in situ Hi-C using the Arima-HiC Kit according to the user guide for mammalian cells. Fragmentation of DNA to obtain an average fragment size of 400 bp was performed with the Bioruptor (Diagenode, 30 cylces, 30 s, on, 90 s off, low). The final Hi-C library was amplified using the KAPA-SYBR Fast DNA amplification reagents (Kapabiosystems) supplemented with EvaGreen (1x) in technical replicates to monitor library amplification. The final sequencing libraries were constructed using the Swift Bioscience protocol provided by Arima. Technical replicates were pooled and cleaned twice using Ampure Beads XP (Beckman, 0.9  beads).