Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: The chromatin samples collected from different mice at the same time point were pooled, frozen in liquid nitrogen and stored at -80˚C. Chromatin was subjected to immunoprecipitation with an anti-POLR2B (Santa Cruz Biotechnology, H-201) antibody as described (Le Martelot et al. 2012). Immuno-precipitated complexes were collected by adsorption to ten µl of protein-A-Sepharose beads that had been pre-blocked with 10 µg/ml of salmon sperm DNA and BSA at 4˚C overnight. The beads were washed with dialysis buffer and ChIP wash buffer, as described. The immuno-precipitated complexes were eluted with 50 mM NaHCO3, 1% SDS, 1mM EDTA and 50 mM of Tris pH8 at 65°C, adjusted to 125 mM NaCl, and incubated at 65°C overnight to reverse the cross-links. After successive treatments with 10 µg/ml of RNase A and 20 µg/ml of proteinase-K, the samples were extracted with a NucleoSpin® Kit (Machery Nagel). The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen). 5-10 ng of DNA was used for the preparation of the library. Libraries for ultra-high throughput sequencing were prepared using Diagenode kit (C05010013) according to the manufacturer's instruction and subjected PE sequencing in a HiSeq 2500. Libraries for ultra-high throughput sequencing were prepared with the using Diagenode kit (C05010013) according to the manufacturer's instruction.