GSM4548040: 17_pH2Ax_6312-CTCFmotifKO_RNP-MycPro; Mus musculus; OTHER
Cells were washed 1X with cut and run wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine), bound to activated ConA beads (Bangs Laboratories BP531), permeabilized in digi buffer (wash buffer + 0.002% digitonin), incubated with antibodies (1 g/CR in digi buffer), washed in digi buffer, incubated with pA-MN (gift from Heinkoff Laboratory, currently EpiCypher 15-1016), and washed in digi buffer. Following the final wash, cells were washed with ice-cold low salt wash buffer (20 mM HEPES, pH 7.5, 0.5 mM spermidine, 0.002% digitonin) and digested using MN-ase digestion buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.002% digitonin) for 25 minutes on ice. Solubilized chromatin was released using an isosmotic stop buffer (170 mM NaCl, 20 mM EGTA, 0.05% Digitonin, 20 µg/ml glycogen, 25 µg/ml RNase A, 2 pg/ml S. cerevisiae fragmented nucleosomal DNA) and was collected using a PCR clean up kit column (EZbioresearch M1001). All antibody incubations and digitonin washing steps were performed for 5 minutes at room temperature and included protease, phosphatase and deacetylase inhibitors (Roche). For library preparation, DNA products were incubated with an end repair master mix (1 X T4 ligation buffer, dNTP, ATP, T4 PNK, T4 DNA Pol and TAQ DNA polymerase) and incubated as follows: 12'C 15 minutes (end polishing), 37'C 15 minutes (5' end phosphorylation) and 58'C 1.5 hour (dA tailing). Polished libraries were purified using AmpureXP beads and ligated to either NEBNext Dual Index (NEB) or Illuminia TruSeq adaptors. Libraries were size selected and enriched by PCR. Finished libraries were sequenced using Illumina HiSeq2500 (Genome Technology Access Center, Washington University).
GEO Accession
GSM4548040