title: PolII-S2P ChIP
organism: Homo sapiens
description: Protocols: U20S AsiSI-ER cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics and 10% FCS (Invitrogen) at 37 C under a humidified atmosphere with 5% CO2. Cells were treated with 300 nM 4OHT for 4 hours ChIP assays were carried out according the protocol described in (Iacovoni et al, 2010). Briefly, 200 ug of chromatin was subjected to immunoprecipitation using 2 ug of a gammaH2AX antibody (Epitomics 2212-1), XRCC4 (Abcam), or Rad51 (Santa Cruz). For ChIP against PolII-S2P (Abcam) 50 ug of chromatin was used, instead of 200 ug. After the washes the beads/chromatin complexes were re-suspended in 200 uL of TE buffer (Tris 10mM pH8, EDTA 0,5mM pH8) and were directly subjected to crosslink reversal in presence of 0,5% SDS overnight at 70 C. Following crosslink reversion, all immunoprecipitated DNA and input DNA were purified by phenol/chloroform, followed by DNA precipitation. DNA amount was quantified using the Quant-iTTM kit from Invitrogen. 10ng of DNA was next used for sequencing.