GSM4767144: WT control, Stat3-IP rep2 [WT-C2]; Mus musculus; ChIP-Seq
Frozen cell pellets were lysed and sonicated in ice to produce DNA fragments of 300-400 base pairs. Residual insoluble chromatin was sedimented by centrifugation at 14,000 rpm for 10 minutes at 4°C. 10% of each supernatant was set aside as the input fraction. For immunoprecipitations, samples were pre-cleared with agarose beads (2h at 4°C), diluted and divided into 2 fractions to which were added either a-Stat3 antibody (124H6 Cell Signalling) or mouse IgG (MFCD00212351, Sigma) as negative control, and incubated on a wheel at 4°C over night. Immune complexes were collected by incubation with protein-A sepharose beads (GE Healthcare) for 1 h and centrifugation. Beads were washed serially in buffers of increasing ionic strength and detergent concentration, complexes were eluted from the beads in bicarbonate and incubated at 65°C for 6 h to reverse crosslinks. Samples were treated with proteinase K and DNA was purified using a GeneJet purification kit. ChIP-Seq libraries were prepared using the NEBNext Ultra DNA library Prep Kit for Illumina (NEB; E7370S) and NEBNext Multiplex Oligos for Illumina (NEB; E7335, E7710, E7730), following instructions in the application note: Low input ChIP-Seq using the NEBNext Ultra Library Prep Kit for Illumina (https://international.neb.com/-/media/catalog/application-notes/low-input-chipseq-using-the-nebnext-ultra-library-prep-kit-for-illumina-e7442.pdf?rev=91023778031446d88ebc26b53d3b8b83). Libraries were sequenced on the Illumina NextSeq 500 using a NextSeq 500 High Output v2.5 150 cycle kit (Illumina; 20024907).