title: GSM5273003: ChIP-seq in immortalized MEFs in hypoxia; Mus musculus; ChIP-Seq
library_construction_protocol: 24x106 MEFs were fixed using a final concentration of 1% paraformaldehyde (PFA) in 200 mM phosphate buffer added to the culture medium and incubated during 10 minutes at 37ºC. Crosslinking reaction was stopped adding 125 mM glycine and incubating during 10 minutes at room temperature. After this, cells were collected from the culture dish with cold PBS with protease inhibitors cocktail (PIC) (Merck) and phenylmethylsulfonyl fluoride (PMFS) (Merck).  Nuclei were extracted and lysed, and chromatin sonicated in a Covaris M220 sonicator to an average of 300bp fragments. Sonicated chromatin was divided in 4 aliquots and incubated overnight at 4ºC with 2µg of antibody (anti-Tfcp2l1, Novus Biologicals; AF5726) per aliquiot. Immunoprecipitated chromatin was then incubated with magnetic beads, washed, TAGmented with Tn5 transposase (Schmidl et al., 2015) and eluted from beads. Then, chromatin was decrosslinked and DNA was purified using Minelute kit (Qiagen) Preparation of ChIPmentation libraries was performed as described for ATAC-seq assays in Buenrostro et al., 2015