GSM3289772: polymerase II 0h ChIA-PET2; Homo sapiens; ChIA-PET
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al (Fullwood et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009, 462(7269):58-64).   BJAB cells were treated with 1.5mM EGS (Pierce Cat # 21565) for 30 min, followed 1% formaldehyde at room temperature for 15 min, and then neutralized using 0.2M glycine.  Chromatin was sheared by sonication, and anti-Pol II monoclonal antibody 8WG16 (Covance, MMS-126R) was used to enrich Pol II-bound fragments.  A portion of ChIP DNA was eluted from antibody-coated beads for quantitation using Picogreen fluorimetry, and for enrichment analysis using quantitative PCR.  Two biological replicate ChIP samples were used for ChIA-PET library construction.