title: GSM3382042: H3S57ph_1 ChIP-Seq HU; Homo sapiens; ChIP-Seq
library_construction_protocol: Native chromatin, digested with MNase was pre-cleared, SDS was added to 0.1% and incubated on ice for 12 min. IP buffer with inhibitors was added to 1x concentration (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100). After storing 10% of the material for Input sample, aliquots were made and H3S57 antibody (8 μg) (each ChIP with approximately 10M cells) and incubated for 14 h with rotation at 4°C. Pre-washed 20 μl beads slurry per sample (Dynabeads protein A) pre-blocked in 5 mg/ml BSA in PBS were incubated and incubated for 2 h. The beads were washed with a total of 3 ml of each: wash A (50 mM Tris, 10 mM EDTA, 75 mM NaCl), wash B (wash A but with 125 mM NaCl), and wash C (wash A but with 175 mM NaCl). Bound nucleosomes were washed one last time with TE buffer containing 50 mM NaCl before being eluted with 300 μl elution buffer. RNAase was added to digest RNA for 2 h and Proteinase K to digest proteins for another 2 h at 37oC. DNA (from IP and Input samples) was precipitated and cleaned-up with phenol/chloroform/isomylalcohol and Qiagen clean-up columns. DNA concentration was estimated by Qubit fluorometry and libraries were prepared using the Illumina's TruSeq ChIP Sample Prep Kit (Low Throughput) according to the manufacture's protocol. Libraries were multiplexed 12 per lane on a HiSeq2500 Illumina sequencing machine and sequenced in paired-end mode using the TruSeq Rapid SBS kit
identifiers.namespace: GEO
identifiers.id: GSM3382042