title: GSM3182471: HH20_HL_WT_CHi-C; Gallus gallus; OTHER
library_construction_protocol: Dissected tissues were dissociated in 10% FCS/PBS with collagenase (C7657, Sigma) to a final concentration of about 1.3 ug/uL, and sample were incubated in Thermomixer at 37°C at 800 rpm for 20 min. After discarding the supernatant, cells were crosslinked with 1% formaldehyde/PBS at RT for 10 min, quenched with glycine and centrifuged to discard the supernatant. Cells were resuspended with PBS containing proteinase inhibitor and then centrifuged again. After removing supernatant, cells were kept at -80°C before use. Hi-C library preparation was performed as described in Belaghzal et al. (Belaghzal et al. 2017) with following changes: (i) Resuspended crosslinked cells in ice-cold Lysis buffer were placed on rotation wheel at 4°C at 30 rpm for 30 min for cell lysis. (ii) For chromatin digestion, 400U DpnII (R0543M, New England Biolabs) was added to the sample and incubated at 37°C at 700 rpm for 4 hrs. Another 400U DpnII was added and sample was incubated overnight. (iii) Blunt-end ligation of biotin filled-in DNA was performed at RT at 30 rpm on rotating wheel for 4 hrs. (iv) No removal of biotin from un-ligated end was performed. (v) DNA was sheared to a size of 200 - 800 bp by using COVARIS E220 with following condition, 175W, 10% Duty factor, 200 Cycles per Burst, 60 sec. (vi) DNA pulldown was done with Dynabeads MyOne Streptavidin T1 (65601, Thermo Fisher). (vii) DNA was measured by Qubit and 200 ng was used for further treatment followed by manufacturer's protocol (SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library).