title: Illumina HiSeq 4000 paired end sequencing; The role of DNA repair endonuclease XPF in transcription
design: The role of DNA repair endonuclease XPF in transcription
library_construction_protocol: For ChIP assay,cells were cross-linked at room temperature for 2.5 min with 1% formaldehyde. Chromatin was prepared and sonicated on ice for 8 min using a Covaris S220 Focused-ultrasonicator. Samples were immunoprecipitated with antibodies (5-6μg) overnight at 4οC followed by incubation for 3 h with protein G Sepharose beads (Millipore) and washed sequentially. Primary  MEFs  were  isolated  from  E13.5  animals  and  cultured  in  standard  medium  containing  Dulbecco's  modified  Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 50μg/ml streptomycin, 50 U/ml penicillin (Sigma) and 2mM L glutamine (Gibco).  : Cells were rinsed with PBS, exposed to tRA (10 μM) (Sigma-Aldrich) and cultured at 37oC for 16h, or to UVC irradiation (10 J/m2) and cultured at 37oC for 2h prior to fixation (2.5 min with 1% formaldehyde, at R.T.). The complexes were eluted and the crosslinking was heat reversed. Purified DNA fragments were analyzed by sequencing. Libraries were prepared by using the Illumina TruSeq DNA nano sample preparation Kit . After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified to create the final libraries. After validation (Agilent 2200 TapeStation) all libraries were individually quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.