GSM5827502: Purified Cardiomyocyte-P0_rep2; Mus musculus; Hi-C
For each biological replicate, 2M purified cardiomyocytes were fixed in PBS supplemented with 1% formaldehyde for 10 min at RT and then quenched with 125 mM glycine for 5 min at RT. Nuclei were isolated by treating with a hypotonic buffer (10 mM Tris-HCl, pH 8.0, 10 mM KCl, 1 mM MgCl2, 0.2% Igepal CA630) on ice for 10 min. Pelleted nuclei were permeabilized using 0.5% SDS and incubated for 10 min at 62 °C. Chromatin was digested using the 4-base cutter restriction enzyme MboI (200 units, NEB) at 37 °C overnight. After inactivation of MboI for 20 min at 62 °C, single stranded overhangs were filled with biotinylated-14-dATP (Life Technologies) and dCTP, dGTP, and dTTP using Klenow DNA polymerase (40 units, NEB) for 90 min at 37 °C. DNA was then ligated for 4 h at 16 °C using T4 DNA ligase (2,000 units, NEB). Reverse crosslinking was performed by incubating at 55 °C with proteinase K for 30 min and then 68 °C overnight. Purified DNA was sonicated using an ultra sonicator to 300-700 bp small fragments. After size selection and purification using SPRI beads (Beckman Coulter), biotinylated DNA was isolated using Dynabeads MyOne T1 Streptavidin beads (Life Technologies). Sequencing libraries were prepared on magnetic beads and final PCR amplification was carried out for 6 cycles. Size distribution of bead-purified libraries was checked using a High Sensitivity D1000 ScreenTape on a Tapestation (Agilent) and quantified using Qubit (Life Technologies). Libraries were sequenced on Hiseq 4000 (100 bp PE, Illumina)