GSM1482893: IP RNAPII control (siScrambled); Homo sapiens; ChIP-Seq
Chromatin immuno-precipitations (ChIPs) were performed as described in Whyte et al. 201228 using ~100 x 106 HeLa-LTR-Luciferase cells as starting material with the exception of an additional nuclei purification step. Briefly, cells were thawed and resuspended in 1ml Buffer A (0.3M SUCROSE, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF), 1ml Buffer B (0.3M SUCROSE, 0.2% NP40, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF) was then added and incubated for 7min on ice, laid over a 8ml cushion of Buffer C (1.2M SUCROSE, 0.2% NP40, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF) and spinned for 20min at 3000rpm on 4  C. Pelleted nuclei were resuspended in 3ml Lysis buffer, incubated for 1 hour, sonicated (Misonix sonicator with the following settings: micro tip, 30sec on, 2min off, amplitude 70, 7min total sonication time). 10ng, as quantified by Qubit   dsDNA HS Assay Kit (Life Technologies), of input and of immuno-precipitated-material was used for library preparation. Librairies were prepared using Illumina ChIP-Seq sample prep kit (non-multiplexed libraries) or TruSeq ChIP Sample Prep Kit (multiplexed libraries) according to manufacturer’s instructions. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. Data quality was assessed using fastqc from the Babraham Institute and the Illumina software SAV (Sequence Analysis Viewer). De-multiplexing and alignment were performed using Illumina's sequencing analysis software (CASAVA 1.8.2).
GEO Accession
GSM1482893