title: GSM4671824: ProB(+)_1%input-rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Ten million of ProB(+) 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 nuclei 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 SFX150 (Branson) for 6 cycles of 20sec sonication followed by 1min rest, with 30% amplitude.  Six ug of anti-Lmo2 Abs (a mixture of 2 ug of NB110-78626 (Novus), 2 ug of ab91652 (Abcam) and 2 ug of AF2726 (R&D systems)) were pre-bound to Dynabeads Protein A/G (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 ug/ml proteinase K).  Precipitated chromatin fragments were cleaned up using PCR purification Kit (Qiagen).  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.
identifiers.namespace: GEO
identifiers.id: GSM4671824