ChIP-seq on human cell lines with anti-PGBD5 polyclonal antibodies

The DNA binding profile of PGBD5 to human chromosomal DNA was done using HEK293-T, HeLa, H4, 8MG, T98G and HCT116 cell lines. Cell lineages were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), at 37 °C and with 5 % CO2. Murine pre-immune and anti-PGBD5 polyclonal sera were produced by DNA vaccination using ICANtibodiesTM technology (In Cell Art, Nantes, France) as described [doi: 10.1007/s00438-013-0754-8]. Chromatin samples prepared from non-synchronous and exponentially growing cells, shearing of the chromatin with Bioruptor apparatus, chromatin immunoprecipitation with purified pA, and purification of populations of immunoprecipitated DNA fragments were done using the iDeal ChIP-seq kit following supplier's recommendations (Diagenode, Ougrée, Belgium). Libraries for Illumina sequencing were made using iDeal ChIP-seq & Library Preparation Kit (Diagenode, Ougrée, Belgium). The monitoring of DNA quantification at various steps of the procedure was carried out with the Qubit® dsDNA HS Assay Kit (=molecular probes, Eugene, USA). The final control of the libraries, the fragment size selection, and the Illumina sequencing (HiSeq 51 nucleotides,TruSeq SBS Kit v3 (Illumina, Fulbourn, United Kingdom) was achieved by the Imagif sequencing platform (CNRS, Gif-sur-Yvette, France) in a way that fulfilled quality recommendations [doi: 10.1371/journal.pcbi.1003326]. Overall, 3 experimental replicates in each cell type were made for the immunoprecipitations done with the pre-immune pA or the anti-PGBD5.