title: GSM8851282: HeLa, Asynchronous, untreated, Rep1, CTCF, ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked using 1% formaldehyde for 10 minutes and then were quenched with 125 mM glycine for 5 minutes at 60 rpm at RT. Cells were washed thrice with icecold PBS, scraped and pelleted down at 2000 rpm at 4°C for 5 minutes. The cell pellet was either stored at -80°C or directly used in further steps. First, the cells were lysed with L1A buffer (10 mM HEPES/KOH pH 7.9, 95 mM KCl, 1 mM EDTA pH 8 and 1% NP-40) for 10 minutes on ice and centrifuge at 3500 rpm for 5 minutes. Nuclei were obtained by adding a nuclear lysis buffer (L2) (50 mM Tris-HCl pH 7.4, 1% SDS and 10 mM EDTA pH 8) supplemented with 1x PIC, to the pellet for 10 minutes on ice. The chromatin was fragmented to an average size of 400-500 bp by 22 cycles of 30 seconds ON and 30 seconds OFF pulses using a Diagenode Bioruptor sonicator. Fragment size was 27 visualised by running the chromatin on 1% Agarose gel. The DNA concentration was checked in NanoDrop. Chromatin lysate was cleared by centrifugation at 12000 rpm at 4°C for 12 minutes. One hundred µg of chromatin lysate was diluted in the Dilution buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 2 mM EDTA pH 8 and 0.5% Triton X-100) in a 1:1.5 ratio and 10% of chromatin lysate was taken as input. The antibody was added and incubated overnight at 4°C with a constant rotation. Antibodies used are: CTCF (D31H2: Cell Signaling Technology, 1µg), RAD21 (ab992: Abcam, 1µg), MRE11 (ab208020: Abcam, 2µg), γH2AX (ab81299: Abcam, 2µg), H2AX (ab :Abcam, 2ug,), ATM (ab : Abcam, 2ug), FANCD2 (NB180, Novus Biologicals, 2ug) and RAD51 (ab176458: Abcam, 2µg). The next day, Protein A/G Dynabeads  which were blocked in 5% BSA for 1 hour, were added (15µl per tube) for 4 hours at 4°C at 12 rpm. This was followed by washes with wash buffer I (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 2 mM EDTA pH 8 and 1% Triton X-100), wash buffer II (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 2 mM EDTA pH 8 and 1% Triton X-100), wash buffer III (20 mM Tris-HCl pH 7.4, 250 mM LiCl, 1% NP40, 1 mM EDTA pH 8 and 10% Sodium deoxycholate) and 1xTE buffer (10 mM Tris pH 8, 1 mM EDTA pH 8) in sequence for 5 minutes at 4°C. Flow through was discarded after each wash step. Then the proteinDNA complex was eluted in 200µl of Elution Buffer (100 mM NaHCO3, 1% SDS) at 37°C. Decrosslinking was performed by adding 14 µl of 5M NaCl to the eluate at 65°C overnight. The DNA was purified using the Phenol:Chloroform:IAA method followed by ethanol precipitation. Finally, the pellet was dissolved in water and the extracted DNA was used in a qPCR reaction to verify the ChIP and proceeded for sequencing library preparation was done using NEBNext Ultra II DNA library Prep kit with Sample Purification Beads (E7103L).