title: GSM5501411: E10.5 trunk Del(CBS1-5) ChIPM CTCF rep2; Mus musculus; OTHER
library_construction_protocol: ChIP-M experiments for CTCF and RAD21 were performed according to (Darbellay et al., 2019a). Briefly, trunk regions of E10.5 embryos (from below the post-occipital region) derived from trans-heterozygous crosses were individually dissociated into single cells with collagenase and fixed in 1% formaldehyde solution for 10 minutes at room temperature. The crosslinking reaction was stopped adding Glycine to a final concentration of 0,125 M and the cell pellet was washed three times with cold PBS with protease inhibitors (Complete mini EDTA –free proteinase inhibitor cocktail; Roche). Fixed samples were stored at -80C. Yolk sacs were used for genotyping. Samples from homozygous mutant embryos or control littermates were resuspended in sonication buffer (Tris HCl pH=8.0 50mM; EDTA 10 mM SDSS 0,25%; protease inhibitors) and the chromatin was sheared in a Covaris S200 sonicator to an average fragment size of 250-300 bp. The sonicated chromatin was diluted 2.5 times with dilution buffer (HEPES pH=7.3 20mM; EDTA 1mM; NP40 1%; NaCL 150mM; protease inhibitors). Antibodies against CTCF (Active Motif #61311; 4gr) or RAD21 (Abcam # ab992; 5ugr) were conjugated with Dynabeads Protein G (Thermofisher) magnetic beads. The bead-antibodies complexes were added to the diluted chromatin and incubated overnight at 4oC in a rotating platform. The day after, chromatin-antibody complexes were washed using a magnetic stand with RIPA buffer (Tris HCl pH=8.0 10mM; EDTA 1mM; Sodium Deoxycholate 0,1% TritonX-100 1%; NaCl 140mM; two washes); RIPA-High salt buffer (Tris HCl pH=8.0 10mM; EDTA 1mM;  Sodium Deoxycholate 0,1% TritonX-100 1%; NaCl 500mM; two washes), LiCl-buffer; (Tris HCl pH=8.0 10mM; EDTA 1mM;  LiCl 250mM; Sodium Deoxycholate 0,5% NP40 0,5%; two washes) and Tris HCl 10mm pH=8.0 (two washes). The chromatin was then tagmented for two minutes with the Tn5 transposase (Illumina). Tagmented chromatin was eluted, reverse crosslinked and purified using Qiagen Minielute columns. Libraries were quantified using the Kapa library quantification kit and PCR amplified using barcoded primers. ChIP-M libraries were pair-end sequenced in an NextSeq 500 sequencer (PE 2x 37-43bp). Nextera library construction were done following the published ChIPmentation protocol (Schmidl, 2015)