title: GSM4307084: IAPEz_ChIRP; Mus musculus; OTHER
library_construction_protocol: E14 ES cells were crosslinked with 3% formaldehyde for 30 min at room temperature. Crosslinking was then quenched with 0.125 M glycine for 5 min. Chromatin was then solubilized by sonicating in lysis buffer (50 mM Tris 7.0, 10 mM EDTA, 1% SDS, 0.5 mM DTT, and RNase Inhibitors). Chromatin is diluted in two times volume of hybridization buffer (750mM NaCl, 1% SDS, 50 mM Tris 7.0, 1 mM EDTA, 15% Formamide, 0.5 mM DTT, and RNase Inhibitors). 10 pmol probes were added to 3 ml of diluted chromatin, which was mixed by end-to-end rotation at 37 °C for 6 hr. Streptavidin beads were washed three times in nuclear lysis buffer and added into the reaction, which was mixed for another 45 min at 37 °C. Beads were captured by magnets (Invitrogen) and washed five times with wash buffer (2x SSC, 0.5% SDS, add DTT and PMSF fresh). chromatin was reverse crosslinked at 65 °C overnight. After 1 hour of RNase A (1unit/μl) at 37 °C and Proteinase K (1unit/μl) digestion at 55 °C, DNA samples were then purified using PCR extraction kit (QIAGEN #28006). The precipitated DNA samples were prepared for DNA deep sequencing according to manufacturer's guidelines (SWIFT, #21096). The precipitated DNA samples were prepared for DNA deep sequencing according to manufacturer's guidelines (SWIFT, #21096).