GSM2995081: NS5 Mock Input, Rep2; Homo sapiens; ChIP-Seq
Cells were scraped, washed two times with cold PBS, and lysed in Cell Lysis Buffer (5mM HEPES-KOH pH 7.9, 85 mM KCl, 0.5% IGEPAL CA-630, 0.5 mM phenylmethane sulfonyl fluoride and cOmplete Protease Inhibitor Cocktail (Roche)). Nuclei were pelleted at 600 g for 10 minutes and lysed with Nuclei Lysis Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethane sulfonyl fluoride and cOmplete Protease Inhibitor Cocktail (Roche)). Chromatin shearing by water bath sonication (Diagenode Bioruptor) was optimized to produce ~200-300 nucleotide fragments, and protein concentration was quantified by Bradford Assay (Thermo Fisher). Chromatin was immunoprecipated overnight at 4C using 500g chromatin and 5g of anti-LEO1 antibody. To purifiy antibody-bound chromatin, 50L of Protein A Dynabeads (Thermo Fisher) were added and incubated at 4C for one hour. Dynabead-bound ChIP samples were washed two times with ChIP Dilution Buffer (50mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100), two times with ChIP High Salt Dilution Buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA and 1% Triton X-100), and one time with LiCl Wash Buffer (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630 and 0.5% sodium deoxycholate) for five minutes each before being eluted. Samples were reverse crosslinked at 65C overnight and treated with RNAseA (Fermentas) and ProteinaseK (Fermentas). DNA was purified by phenol-chloroform extraction and ethanol precipitation. Library preparation for next generation sequencing was done using NextFLEX ChIP-seq Library Preparation Kit (BioO Scientific) according to manufacturer recommendations.
GEO Accession
GSM2995081