title: GSM5981810: LP_aCbfa2t3_ChIP_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Ten million of LP cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by an additional 10 min with addition of formaldehyde up to 1 %.  The reaction was quenched by addition of 1/10 volume of 0.125 M 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 a Bioruptor (Diagenode) for 18 cycles of 30 sec. sonication followed by 30 sec. rest, with high power.  Five ug of anti-Zbtb1 Abs (a mixture of 2.5 ug of S303-242A (Bethyl) and 2.5 ug of ab79455 (Abcam)) or anti-Cbfa2t3 (a mixture of 2.5 ug of 17190-1-AP (Proteintech) and 2.5 ug of sc-373691 (Santa Cruz)) were pre-bound to Dynabeads anti-Rabbit Ig or Protein A/G (Invitrogen) and then added to the diluted chromatin complexes.  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 ChIP DNA Clean & Concentrator (Zymo).  ChIP-seq libraries were constructed using NEBNext Ultra II DNA Library Prep with Sample Purification Beads (E7103S, NEB) and NEBNext Multiplex Oligos for Illumina (E7500S, NEB), and sequenced on Illumina NextSeq500 in single read mode with the read length of 75 nt.