GSM8499178: GCT72-FOXA2_II; Homo sapiens; ChIP-Seq
Upon two washing steps with PBS, DNA-protein-crosslinking was performed by adding 80 µl of 'ChIP cross-link Gold' (Diagenode, Seraing, Belgium) to 20 ml PBS containing 1 mM MgCl2 for 30 min. Subsequently, cells were washed twice with PBS before continuing the ChIP with another crosslinking-step using formaldehyde (Sigma-Aldrich, Taufkirchen, Germany) and using the 'SimpleChIP Plus Enzymatic Chromatin IP Kit' (Cell Signaling Technology, Leiden, The Netherlands) according to the manufacturers protocol. To digest and fragmentize the chromatin, 0.5 µl micrococcal nuclease per IP was added and the samples were sonicated three times for 20 sec followed by a pause of 30 sec at an amplitude of 80 %. At that step, one IP of the quadruplicates was used to purify DNA and to validate the chromatin digestion and concentration as a quality control. Upon successful digestion and fragmentation, 2 % of input control was extracted and the ChIP was continued according to the manufacturers protocol by incubating 10 µg of antibody / IP over night at 4°C on a Hula mixer at 20 rpm. Upon immunoprecipitation using 'Protein G' magnetic beads, the chromatin was eluted and reversed cross-linked before purification of DNA using spin columns. ChIP-seq was performed of IP samples (GCT72, 1411H; each n = 3) and input controls (n = 2) according to the standard procedure at Novogene Corporation (Munich, Germany). Briefly, the quantity and quality of purified DNA was measured by using the 'Qubit DNA Assay Kit' in Qubit 3.0 Fluorometer (Life Technologies, CA, USA) and the NanoPhotometer spectrophotometer (IMPLEN, CA, USA), respectively. All samples required a minimum of 5 ng purified DNA, which indicated main peaks between 100 and 500 bp on an agarose gel. The library was constructed by Novogene Corporation and its quality was monitored on the Agilent Bioanalyzer 2100 system before being pair-end sequenced on an Illumina platform (Illumina, CA, USA).