GSM5741895: GM12282_REF_CaptureC_rep1; Homo sapiens; OTHER
20M GM12282 (homozygous REF) or GM11931 (homozygous ALT) cells were fixed with 2% PFA in PBS at RT for 10min. Fixation was stopped by adding glycine at a final concentration of 125 mM and incubated for 5 min at RT under rotation. Cells were then washed twice with ice cold PBS, resuspended in lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% NP40 and supplemented 1x cOmplete protease inhibitor (Roche)), incubated 20 min on ice and snapped frozen with the buffer. The Capture-C was performed as described in Davies J. et al., 2015 with the following modifications: NlaIII restriction enzyme (New England Biolabs) was used and incubated for 24 h. Ligation was performed for 22 h. 5 μg of DNA (3C library) in 120 μl TE were sonicated using a Covaris E220 with the following settings: 180 s, 10% duty factor, 175 W of intensity, 200 bursts/cycle with the #500141 intensifier (Covaris). 1 μg of sonicated DNA was used to prepare the library using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) as described by the manufacturer. After adapter ligation, DNA was clean-up using 1.8X AMPure X beads (Beckman) and amplified with Herculase II (Agilent) for 6 cycles. Each biological replicate (3 biological replicate per cell line) was labelled with a different barcode and all samples (a total of 6) were pooled together (2 μg of each). For the first capture, 2.9 nM of each biotinylated probes (Supplementary Table 5) was used and incubated for a total of 72 h. The pooled captured library was amplified for 14 cycles and a second capture was performed with all resulting DNA (130 ng) and 2.9 nM of each biotinylated probes for 24 h. The pooled library was further amplified for 14 additional cycles. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).