GSM2107747: input DNA for ChIP on thymoctes fixed with formaldehyde#1; Mus musculus; ChIP-Seq
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (NEB #E6240) following manufacturer’s instructions. Briefly, ChIP DNA was purified and end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3’ 'A' base for ligation of NEBNext Multiplex Oligos for Illumina (NEB #E7335) which have a single 3' overhanging 'T' base and a hairpin structure. After ligation, adapters were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified for 18 cycles followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067). Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions.