title: GSM7310687: HaCaT_Tet-on-Ikzf1, DoxD3, CTCF; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed in 1% of fresh formaldehyde for 10 minutes at room temperature, quenched with glycine and washed twice with ice-cold PBS. Cells were re-suspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 minutes at 4°C, pelleted, and then re-suspended in lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH8) for 5 minutes at 4°C. Cells were finally re-suspended in RIPA buffer (50mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Nadeoxycholate, 0.2% SDS, 0.5 mM PMSF, and 1x Protease inhibitor cocktail (Roche)) to a final concentration of 5x107 cells/ml. Chromatin was sonicated to an average size of 350bp with a Branson Sonifier 450 equipped with a micro probe. Chromatin was cleared by centrifugation at 20,000xg for 10 minutes, and then incubated with 5-10μg of antibodies pre-bound to Dynabeads protein G (Life technologies) and rotated overnight at 4°C. After extensive washing on beads, bound chromatin was eluted and de-crosslinked in 300μl of elution buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, 0.3M NaCl) overnight at 65°C. Proteinases K was added to a final concentration of 200 μg/ml and incubated at 45°C for 2 hours. DNA was ethanol precipitated, re-suspended in TE and purified using the DNA Clean & Concentrator-5 kit (Zymo Research). Purified ChIP DNA was end repaired, end adenylated, and then ligated with Illumina Truseq indexed adaptors. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and then amplified with KAPA HiFi DNA Polymerase (KAPA Biosystems) for 8 to 13 cycles. After amplification, the library DNA was size selected with AMPurex XP beads to 200-600 bp range, and the purified libraries were multiplexed for sequencing in house with an Illumina NextSeq 550 system.