GSM2533833: HiC_FACS; Mus musculus; OTHER
HiC and library preparation was carried out using the in-situ method as described previously (Rao et. al., 2014) with minor modifications. In order to maximize library complexity, FACS-purified samples were split into batches of 1x10^6 cells and processed separately. In brief, cells were digested overnight at 37C using 500U of DpnII. After biotin filling, proximity ligation was carried out for 4 hours at 18C with 2000U T4 DNA Ligase. After reverse-crosslinking, DNA was purified using ethanol precipitation and sheared to 300-400bp fragments using Covaris S220 sonicator. Ligation fragments containing biotin were immobilized on MyOne Streptavidin T1 beads (ThermoFished Cat.N: 65602), end-repaired and a-tailed as described. NEXTflex adaptors (Bioo Scientific, Cat.N: 514101) were then ligated and fragments were PCR amplified using KAPA HiFi Library Ampification Kit (Kapa Biosystems, Cat.N: KK2620) for 6-8 cycles. DNA was then double-size selected using AMPure XP beads (Agencourt, Cat.N: A63881) in order to isolate fragments between 300 and 800bp.