GSM2805037: ctrl_1 ChIP-Seq; Mus musculus; ChIP-Seq
mES cultured in GS underwent cross-linking with 1% formaldehyde (Applichem) and quenching with 10X Glycine (Millipore) as well as nuclear extraction. Then, chromatin solution was sheared by Bioruptor® Plus (Diagenode) to obtain about 200bp fragments for Sequencing. Immunoprecipitation protocol was performed after overnight incubation at 4°C with 8 μg of Zeb1 (H-102 from Santa Cruz) antibody with Magna-ChIP™ A/G (Millipore) kit according to manufacturer's instruction. DNA fragments were recovered and analysed by Sequencing. ChIP samples were processed for sequencing on the Ion Torrent platform using a modified DNA Library protocol based on the Ion Xpress Plus Fragment Library Kit (Thermo Fisher). 1-10ng dsDNA were used as starting material. After end repair of DNA fragments, Ion Torrent specific and barcoded DNA, Adapters were ligated and covalently bound by nick translation, followed by amplification with 18 cycles using the components of the Ion Xpress Plus Fragment Library Kit. The final SPRI bead cleanup was performed as double size selection with 0.7x DNA/Bead ratio followed by 0.8x DNA/bead ratio. QC of obtained libraries was done on Bioanalyzer 2100 (Agilent) and quantitation was measured on Qubit 2.0 with HS DNA Assay. Libraries were diluted to a final concentration of 10 pM and used in equimolar ratios for template preparation on Ion Touch 2 instrument. Libraries were pooled on PI Ion Torrent V2 Chips to obtain a minimum amount of 20M raw reads per sample.