title: GSM2356842: input DNA; Mus musculus; ChIP-Seq
library_construction_protocol: Embryonic  NSCs  (approx.  10  x  10^6 cells)  were  fixed  with  1.5  mM  ethylene  glycol bis[succinimidyl  succinate]  in  DMSO  for  20  min  at  RT, followed  by  adding formaldehyde  to  a  final  concentration  of  1%,  incubated  for  10  min  at  RT  and  finally quenched by 0.2 M glycine. Cells were then lysed and sonicated with a microtip for 10 seconds  followed  by  a  1  min  break  for  10  cycles.  After  adding  1%  Triton-X  to  the sonicated lysates, ChIP was performed overnight at 4°C with Dynabeads M-280 Sheep anti-Rabbit IgG (Thermo Fisher, 11203D) linked to rabbit anti-E2A (1.5 µg, sc-349X, Santa Cruz Biotechnology). 10% of sonicated suspension was kept as input control. After washing and  elution  of  chromatin-antibody  complexes  from  the  beads  at  65°C  overnight, crosslinking  was  reversed  by  proteinase  K  and  RNase  digestion.  Genomic  DNA  and DNA  from  the  sonicated  input  were  purified  using  a  PCR  Purification  Kit  (Qiagen). Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).