title: GSM4495663: Input replicate 1. BxPC3; Homo sapiens; ChIP-Seq
library_construction_protocol: For isolation of chromatin, cells were lysed with lysis buffer (10 mM HEPES pH 7.4, 10 mM KCl, 0.05% NP-40) with protease inhibitor cocktail (Roche) for 30 minutes and nuclei were collected by centrifugation at 600 g for 10 minutes. Nuclear lysis was performed with low salt buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl2, 1% TritonTM-X100) with protease inhibitor cocktail for 30 minutes and chromatin was collected by centrifugation at 5000 g for 10 minutes. Chromatin was resuspended in NP-40 lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 1% NP-40) and sonicated with S220 Focused Ultrasonicator (Covaris) with settings: incident peak power: 140, duty cycle: 5%, burst/cycle: 200 for optimal chromatin size of 200 to 500bp in length. Cell debris was then pelleted by centrifugation at 8000 g for 10 minutes at 4°C. 50ng of each biological replicate was used for library preparation using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England BiolabsE7370S), and amplified using 10 cycles of PCR using NEBNext Multiplex Oligos for Illumina (New England Biolabs E7335S andE7500S). DNA was size-selected using Agencourt AmpureXP beads (Beckman Coulter A63881), following the protocol in the NEBNext Ultra kit for a 200-bp to 600bp average insert size. Library fragment size distribution was assessed on a Bioanalyzer 2100 instrument (Agilent Technologies), using the DNA 1000 kit (Agilent Technologies 5067-1504). Quantification of libraries was performed using the NEBNext Quant Kit for Illumina® (NEB E7630L).
identifiers.namespace: GEO
identifiers.id: GSM4495663