GSM8395125: FLAG_CTCF_R377C_ID_rep2; Mus musculus; ChIP-Seq
mESCs were dissociated using TrypLE and and double cross linked using 25mM EGS (ethylene glycol bis(succinimidyl succinate)) and 1% formaldehyde. Fixed cells (10 million) were resuspended in 350 μl ice cold lysis buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.1% sodium deoxycholate, 0.5% N-lauroysarcosine and 1X protease inhibitors) and lysed for 10 min by rotating at 4° C. Chromatin was sheared using a bioruptor (Diagenode) for 15 minutes ( 30 sec on, 30 sec off, high output level). FLAG M2 Magnetic Beads were used for FLAG immunoprecipitation. In other cases ( Cohesin, IgG) antibodies were bound to protein A magnetic beads by incubation on a rotator for one hour at room temperature. Samples were washed and tagmentation were performed as per the original ChIPmentation protocol (Schmidl et al., 2015) using 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina #20034198 ). Chromatin was then eluted and de-crosslinked and DNA was purified using MinElute Reaction Cleanup Kit. Purified DNA was amplified as per the ChIPmentation protocol (Schmidl, Rendeiro et al. 2015) using indexed and non-indexed primers and NEBNext High-Fidelity 2X PCR Master Mix. Library quality and quantity were estimatred using Tapestation and were then sequenced with the Novaseq6000 Illumina technology according to the standard protocols and with around 200 million 150bp paired-end total per sample.