Cells (10^6 per ChIP) were cross-linked in suspension for 10 min in media containing 1% formaldehyde before quenching with 1.25mM glycine. The cells were washed twice with ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1g/ml aprotinin and 1g/ml pepstatin A), harvested and treated with SDS lysis buffer for 10 min on ice. Chromatin lysate was sonicated to generate a majority of fragments between 200 and 500bp. 10µg of each antibody was used per ChIP. No antibody controls were also included, and no precipitation was observed by quantitative Real-Time PCR analysis. Input samples were processed in parallel. The antibody/protein complexes were collected by Protein A/G PLUS agarose beads (Santa Cruz, sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1M NaHCO3, treated with proteinase K for 1 hour, followed by DNA purification using phenol/chloroform extraction and ethanol precipitation, and finally resuspended in 30ul H2O. The ChIP DNA was quantitated using Qubit and 10ng was used for Illumina library preparation using Illumina ChIP-seq DNA Sample Prep Kit. Libraries were prepared using the Illumina TruSeq Chip Library Prep Kit. The resulting libraries were sequenced on the Illumina HiSeq 2000 or 2500 platform configured for 50bp single-end reads.
GEO Accession
GSM2610547