GSM5456173: MCF-7 cells - Input_Rep2; Homo sapiens; ChIP-Seq
Briefly, 20 million cells were subjected to fixation with 16% formaldehyde prior to cytoplasmic and nuclear lysis. Nuclear lysates were sonicated for 33 cycles (30 sec on/30 sec off) and precleared with Dynabeads Protein G. Precleared lysates were incubated overnight with 6 micrograms of ChIP antibody, after which antibody-DNA complexes were captured onto Dynabeads Protein G for 4 hours. The beads were washed several times in a series of lysis buffers prior to elution and Proteinase K treatment. ChIP DNA was purified using a phenol chloroform isoamyl alcohol extraction method. 1ng of input or ChIP DNA were used to generate Illumina-compatible indexed libraries using a MicroPlex Library Preparation Kit v2 (Diagenode) following the manufacturer's instructions. Libraries were size-selected to an average fragment size of 300-400bp. The ChIP library pool was sequenced with an Illumina Next Seq 500 High Output Kit for 75 cycles in single-end mode.