title: GSM3656412: ChIP_combined; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP protocols were modified from previously described (Shen et al., 2009). DNA from two replicates was mixed and sent for library construction and sequencing. ChIP samples were quantified using a Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and qualified by Agilent Bioanalyzer 2100(Agilent Technologies, Palo Alto, CA, USA). Next generation sequencing library preparations were constructed following the manufacturer's protocol (NEBNext® Ultra™II DNA Library Prep Kit for Illumina®). For each sample, at least 10 ng ChIP product was used for library preparation. The ChIP product was treated with End Prep Enzyme Mix for end repairing, 5' Phosphorylation and dA-tailing in one reaction, followed by ligation to adaptors with a “T” base overhang. Adaptor-ligated DNA was then recovered using AxyPrep Mag PCR Clean-up (Axygen). Each sample was then amplified by PCR for 8 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up, validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 3.0 Fluorometer. Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer's instructions (Illumina, San Diego, CA, USA).