GSM1153764
GSM1153764: input DNA; Homo sapiens; ChIP-Seq
Following cross-linking, glycine was added to a final concentration of 1.25 M and cells were washed in PBS and lysed in 300 ul of cell lysis buffer (85 mM KCl, 0.5% NP-40, 5 mM PIPES pH 8.0, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)) per 10 7 cells. Following a 10 minute incubation on ice, cell nuclei were pelleted by centrifugation at 8000 rpm for 5 minutes at 4 oC in a bench-top Microfuge. The nuclei were then resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0 plus protease inhibitors) per 10 7 cells and sonicated to reduce DNA length to between 200 and 600 bp. ChIP was carried out using 600 µl of chromatin from 30 x 10 6 cross-linked cells diluted 10-fold in IP dilution buffer and pre-cleared with protein A sepharose beads. An input control sample was removed and EBNA 3 proteins precipitated overnight with rotation at 4ºC using 60 µl sheep polyclonal anti-EBNA 3 antibodies (Abcam, ab16128). EBNA 2 was precipitated using 48 µg EBNA 2-specific mouse monoclonal antibody (PE2) followed by an additional 2 hr incubation with 80 µg rabbit anti-mouse antibodies (Dako). BSA-coated protein A sepharose beads were then added and immune complexes collected by rotation at 4ºC for 3 hrs. Beads were washed for 10 minutes at 4 ºC with rotation using a series of different wash buffers. Complexes were washed once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl), once in LiCl wash buffer (250 mM LiCl, 1% NP40, 1% Na deoxycholate, 1 mM EDTA, 10 mM Tris pH 8.0) and twice in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Immune complexes were eluted in TE elution buffer (10 mM Tris pH 8.0, 5 mM EDTA, 1% SDS) at 65 ºC for 15 minutes. TE elution buffer was added to the input control sample and all samples were then incubated at 65 ºC overnight to reverse the crosslinks and samples were treated with 0.2 µg/ml RNAse A for 1 hr at 37 ºC to remove RNA. DNA was purified using QIAquick gel extraction (Qiagen). ChIP and input DNA (10 ng) was used to generate sequencing libraries with a ChIP-seq sample prep kit (Illumina). DNA fragments were end repaired, phosphorylated, 3'-dA overhangs added and adapters ligated according to the manufacturer's instructions. PCR-amplified samples (16-cycles) were separated on a 2% agarose gel in TAE buffer, visualized using SYBR-safe stain and a Dark Reader transilluminator (Clare Chemical Research). The region of the gel containing 150-350 bp DNA fragments was excised and DNA purified using a gel extraction kit (Qiagen). The library was quantified using an Agilent bioanalzer and subjected to 35bp single-end read sequencing with an Illumina Genome Analyzer IIx.
GEO
GSM1153764
GEO Accession
GSM1153764