GSM3336255: CENP-A TAP G1 input; Homo sapiens; ChIP-Seq
TAP- or LAP -tagged chromatin were purified in two steps. In the first step native TAP-tagged chromatin was immunoprecipitated by incubating the bulk soluble mononucleosome pool with rabbit IgG (Sigma-Aldrich) coupled to Dynabeads M-270 Epoxy (Thermo Fisher Scientific, 14301). Alternatively, CENP-ALAP chromatin was immunoprecipitated using mouse anti-GFP antibody (clones 19C8 and 19F7, Monoclonal Antibody Core Facility at Memorial Sloan-Kettering Cancer Center, New York) (Heiman et al., 2008) coupled to Dynabeads M-270 Epoxy. Chromatin extracts were incubated with antibody bound beads for 16 h at 4 ÅãC. Bound complexes were washed once in buffer A (20 mM HEPES at pH 7.7, 20 mM KCl, 0.4 mM EDTA and 0.4 mM DTT), once in buffer A with 300 mM KCl and finally twice in buffer A with 300 mM KCl, 1 mM DTT and 0.1% Tween 20. In the second step, TAP-chromatin complexes were incubated 16 h in final wash buffer with 50μl recombinant TEV protease, resulting in cleavage of the TAP tag and elution of the chromatin complexes from the beads. Alternatively, CENP-ALAP chromatin was eluted from the beads by cleaving the LAP tag using PreScission protease (4 h, 4ÅãC). For CENP-ATAP co-IP experiments, bound complexes were washed and proteins associating with CENP-ATAP were denatured by boiling in sample buffer, run on SDS-PAGE and blotted for CAF1 subunits and MCM2. ChIP libraries were prepared following Illumina protocols with minor modifications (Illumina, San Diego, CA). To reduce biases induced by PCR amplification of a repetitive region, libraries were prepared from 80-100 ng of input or ChIP DNA. The DNA was end-repaired and A-tailed and Illumina Truseq adaptors were ligated. Libraries were run on a 2% agarose gel. Since the chromatin was digested to mononucleosomes, following adaptors ligation the libraries size was 250-280 bp. The libraries were size selected for 200-375 bp. The libraries were then PCR-amplified using only 5-6 PCR cycles since the starting DNA amount was high. Resulting libraries were sequenced using 100 bp, paired-end sequencing on a HiSeq 2000 instrument per manufacturer's instructions with some modifications (Illumina, San Diego, CA).
GEO Accession
GSM3336255