title: GSM5209365: ChIP-seq against CTCF in ES-cells clone with the ALL genotype; Mus musculus; ChIP-Seq
library_construction_protocol: Fixed nuclei were extracted adding 5 mL of ice-cold custom-made lysis buffer containing the following components: 10mM Tris HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, Protease Inhibitor in Milliwater. Extracted nuclei were then collected, snap frozen and stored at -80 °C or further processed using iDeal ChIP for Transcriptional Factors (Diagenode) (Cat. N. C01010055). Chromatin was shared using Diagenode Bioruptor in order to get a fragment size of 200-500 bp. Immunoprecipitation was done using 15-20 mg of chromatin and 1 μg of CTCF Ab (Diagenode:C15410210). ChIP-seq libraries were prepared using the NEBNext Ultra II Library Prep Kit for Illumina. Library prep was entered with 500pg-1μg of immunoprecipited DNA and processed according to the kit guidelines (NEBNext End Prep, Adaptor Ligation, PCR enrichment of Adaptor-Ligated DNA using NEBNext Multiplex Oligos for Illumina). Clean up and size selection with AMPure beads (NEB). Library was sequenced with single end read of 75 nt.