title: GSM1481686: HeLa_X-ray_3h_H3_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin collection was performed as previously described (Angrisano et al., 2011), with minor adjustments. HeLa cells were fixed with 1% formaldehyde for 10 minutes at room temperature (RT) and cross-linker was quenched by the addition of 0.125 M glycine (5 minutes at RT). Nuclei were isolated after mild lysis in hypotonic buffer (10 mM Hepes pH 8, 1.5 mM MgCl2, 60 mM KCl) and 20 strokes in a tight dounce homogenizer. Chromatin was sheared in sonication buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1). Fragmentation of chromatin was carried out by ultrasound treatment (Bioruptor™ UCD200) so that fragments of 200-300 bp length were produced. 60-120 µg chromatin were immunoprecipitated with anti-H2AX (A300-83A, Bethyl Laboratories, 3 g), anti-gH2AX (Clone JBW301, Upstate, 5 g) and anti-H3 (ab1791, Abcam, 3 g) antibodies. The collected chromatin (ChIP sample) was then reverse crosslinked in the presence of 200 mM NaCl at 65°C for at least five hours, followed by RNase A (50 µg/mL) treatment for 30 minutes at 37°C and proteinase K (100 µg/mL) treatment for three hours at 50°C. DNA elution was carried out in 1% SDS, 100 mM NaHCO3, in rotary shaker at RT for 15 minutes. Pure DNA was isolated using the Qiagen PCR purification kit and 15 ng of size selected DNA fragments were used to produce ChIP-seq libraries Libraries were generated from 15 ng of immunoprecipitated DNA using the Illumina ChIP-Seq DNA sample Prep. Kit according to the suppliers information.
attributes.tag: GEO Accession
attributes.value: GSM1481686