GSM7596453: Keratinocytes, siCtrl, MTA2-IP, rep3; Homo sapiens; ChIP-Seq
Cells were fixed using 1% formaldehyde at RT for 10 min, the reaction was quenched with 125 mM glycine at RT for 5 min. Cells were pelleted and then lysed in Swelling buffer (100 mM Tris, pH 7.5, 10 mM potassium acetate, 15 mM magnesium acetate, 1% Igepal, 1x cOmplete Protease Inhibitor Cocktail (Roche) and 1 mM AEBSF). Chromatin fragmentation was subsequently performed in RIPA buffer for 30 min using the S220 Focused-ultrasonicator (Covaris) in the Freq sweeping mode, intensity 8, 20% duty cycle and 200 cycles per burst. 100 µg fragmented chromatin underwent IP with 5 µg α-MTA2 antibody (Abcam, ab8106) for 16 h at 4 °C. 40 µl Protein G Dynabeads (Invitrogen) were added to the IP and incubated for 45 min at RT. The beads were washed three times with Wash buffer (100 mM Tris, pH 9.0, 0.5 M lithium chloride, 1% Igepal, 1% sodium deoxycholate and 1 mM AEBSF) followed by the elution with Elution buffer (50 mM sodium bicarbonate and 1% SDS). For reverse cross-linking, 0.2 M sodium chloride was added, and the chromatin incubated at 67 °C for 4 h following a purification step with the QIAquick PCR Purification kit (Qiagen) NEBNext Ultra II DNA Library Preparation kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs) was used following the manufacturer's instructions