title: GSM4471559: Phase2_Rbpj_ChIP-rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq; Five to ten million of Phase1 or Phase2 cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%.  The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco).  Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power.  Five g of anti-Rbpj mAbs (a mixture of 2.5 g of 5313 (CST) and 2.5 g of ab180588 (Abcam)) were pre-bound to Dynabeads anti-Rabbit Ig (Invitrogen) and then added to the diluted chromatin complexes in parallel aliquots. The samples were incubated overnight at 4 °C, then washed and eluted for 6 h at 65 °C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 g/ml proteinase K). Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.
attributes.tag: GEO Accession
attributes.value: GSM4471559