Name: GSM6749200
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: 500.000 to 1.000.000 cells were used for ChIP per sample. Cross-linking was done with 1% formaldehyde in DMEM added directly to cells in culture dishes for 8 min at room temperature. Quenching was performed using 0.1M glycine (final concentration). The cells were washed with ice-cold PBS and scrapped with PBS with protease inhibitor cocktail (Cat. 11697498001, Roche). Lysis was performed in LB1 buffer (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Triton X100, protease inhibitors) by rotating for 10 min at +4C, followed by centrifugation at 2000g for 5 min. The pellet was incubated in LB2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, protease inhibitors) by rotating for 5 min at +4C, followed by centrifugation at 2000g for 5 min. The pellet was resuspended in a buffer containing 50mM Tris-HCl pH8.0, 10mM EDTA, 1% SDS, the sonication was performed for 45 cycles, 30-sec intervals on/off. Debris was removed by centrifugation, and 10% from the sonicated material was saved as input. Prior to immunoprecipitation, 5ug antibody and 100ul Protein A beads were pre-incubated overnight at +4C in a total volume of 250ul, adjusted with PBS with 5mg/ml BSA, followed by three washes using the same solution. Immunoprecipitation was done by combining the sonicated material with the bead-antibody complexes, in a total volume of 400ul adjusted with ChIP dilution buffer (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X100, protease inhibitors), by rotating overnight at +4C. After the incubation, the beads were washed 6 times with RIPA buffer (50mM HEPES-KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% sodium deoxycholate) followed by one wash in TE (10mM Tris-HCl pH8.0, 1mM EDTA). Reverse-crosslinking was done in elution buffer containing 1% SDS, 100mM sodium bicarbonate, and 200mM NaCl at 65C for 9-15 hours. The input sample was also reverse-crosslinked. After this step, the beads were removed from the mixture, and the remaining DNA in the solution was treated with 8 ug RNAse A for 1h at 37C, followed by 80ug proteinase K for 2 hours at 55C. DNA was purified using MinElute columns and then used for ChIP-Seq library preparation using the NEXTflex Rapid DNA-Seq Kit (NOVA-5144-02) according to the manufacturer's protocol with some minor modifications. Briefly, we amplified the final libraries with 4 cycles of PCR, then performed an AMPure XP-based size selection (0.5x to eliminate larger fragments followed by 1.8x to extract the smaller fragments), and then continued with 8 more PCR cycles on the size selected material. The final libraries were then cleaned up with 0.8x vol AMPure XP beads.