title: GSM8745024: CTCF ChIP, ESC, CTCF-ZF1 mutant rescue, rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells in a confluent 15 cm tissue culture plate washed once with PBS and fixed for 10 minutes with 1% formaldehyde in 15 mL fixation buffer (10 mM HEPES pH 7.5, 15 mM NaCl, 0.15 mM EDTA, 0.075 mM EGTA in DMEM). Formaldehyde was quenched with 125 mM glycine, incubated for 5 mins at room temperature. Cells were washed and scraped in PBS and transferred to 15 mL tubes. Nuclei were isolated using buffers supplemented with 1x protease inhibitor cocktail (Roche 11873580001) in the following order: 5 mL LB1 (20 mM Tris pH 7.5, 10 mM NaCl, 1 mM EDTA, 0.2% NP-40; 10 min on ice), 5 mL LB2 (20 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; 10 min on ice), and 1.2 mL LB3 (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Nuclei in LB3 were transferred to 15 mL Bioruptor Pico tubes (Diagenode C30010017) containing 800 mg sonication beads. Chromatin was fragmented in LB3 to an average size of 200-250 bp using a Bioruptor Pico (Diagenode). Insoluble material was pelleted by centrifuging at 15,000 g for 10 minutes in 4oC, and supernatant was kept. Supernatant containing sonicated chromatin were pre-cleared with 25 uL Protein G Dynabeads (Invitrogen 10004D), incubated rotating in 4oC for 1 hour. For each reaction, 2-4 ug antibody was incubated with 10 ul Protein G Dynabeads in LB3, rotating at 4oC for 3 hours. Antibody-bead complexes were added to 300 ug of pre-cleared chromatin and incubated rotating at 4oC overnight. 30 ug chromatin was used as input control. Beads were then washed 4 times with RIPA buffer (50 mM HEPES pH 7.5, 500 mM LiCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40) and washed once with TE50 buffer (10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl). The beads-bound chromatin was eluted in 125 uL elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS), incubated at 65°C for 1 hour. Eluted chromatin was de-crosslinked by incubating overnight at 55°C, shaking 1000 rpm, with 0.6 mg/mL proteinase K and 0.1 mg/mL RNase A. 1x volume Phenol:Chloroform:Isoamyl Alcohol (25:24:1) solution was added to the elution and aqueous phase was isolated after centrifugation. DNA was purified from the aqueous phase using a PCR purification kit (Zymo Research DCC-5 D4013) following manufacturer's instructions. Libraries were prepared using NEBNext Ultra II DNA Library prep kit for Illumina (E7645S) following manufacturer's instructions but using ½ volume for all the reactions. NEBNext Multiplex Oligos for Illumina® (Dual Index Primers Set 1, E7600S) were used for barcoding.