title: GSM8372172: es_c5-c6d_rep4; Mus musculus; Hi-C
library_construction_protocol: To stop fixations, Glycine was added at final concentration of 0.13M and incubated for 5 minutes at RT and 15 minutes on ice. Cells were then washed once in cold PBS, centrifuged at 2500g 4oC for 5 mins (these centrifugation conditions were used for all washes following fixation) and pellets frozen at -80oC. Thawed cell pellets were then lysed (10mM Tris-HCL pH8, 10mM NaCl, 0.2% Igepal CA-630, Roche Complete EDTA-free Sigma #11836170001) and digested with 600U of DpnII (NEB).Biotin fill-in was done by incubating cells with a mixture of dCTP, dTTP, dGTP, Klenow polymerase (NEB M0210L) and 37.5µl Biotin-14-dATP (Thermo 19524016) for 4h at RT while shaking at 900rpm for 10 seconds every 5 minutes. Ligation was done overnight at 160C using T4 ligase (NEB cat #M0202M). Sonication was done using Covaris onetube-10 AFA strips using the following parameters for a 300bp fragment size (Duration: 10secs, repeat for 12 times, total time 120 secs, peak power-20W, duty factor 40%, CPB-50).Sonicated material was then size selected using SPRI beads with the following ratios: 0.55X and 0.75X. Hi-C material was then bound to 150µl Streptavidin C1 beads (Thermo 65002), washed and recovered following manufacturers recommendations. Bead-bound DNA was resuspended in 50µl 10mM Tris HCl. Library-prep with material on the beads was done using the Hyper Prep kit (Roche KK8502). For Capture Hi-C capture reactions, 1ug of Hi-C library per sample were hybridized with 120 nucleotide biotinylated RNA probes using the SureSelect kit from Agilent and PCR amplification using the polymerase from the Kapa Hyper Prep Kit. Following washes, material was amplified by PCR using the Kapa polymerase from the the Hyper Prep kit (Roche KK8502). Material from different samples was then combined and 1µg of pooled libraries was recaptured.