ena-STUDY-INRA-25-01-2017-07:39:40:592-158
Diversity of genomic targets of SETMAR isoforms in two colorectal cell lines
The DNA binding profile of certain SETMAR isoforms to human chromosomal DNA was done using two colorectal cell lines, SW48 and HT29. These two lines were chosen because of their expression profiles for SETMAR isoforms, HT29 only expressing the V2 isoform whereas the isoforms V1, V2, X2, V5 and HSMAR1 are present in SW48.Human colorectal cancer cell lineages (SW48 and HT29) 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-HSMAR1 polyclonal sera were produced by DNA vaccination using ICANtibodiesTM technology (In Cell Art, Nantes, France) as described [doi: 10.1007/s00438-013-0754-8]. Polyclonal antibodies (pA) contained in pre-immune and anti-HSMAR1 murine sera were purified using Protein A/G MagBeads (GenScript, Piscataway, USA). Their quality was then verified by PAGE after staining with coomasie blue and their concentration was defined using the BCA Protein quantification Kit (interchim, Monluçon, France).Chromatin samples prepared from non-synchronous and exponentially growing SW48 and HT29 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-HSMAR1 pA.
The DNA binding profile of certain SETMAR isoforms to human chromosomal DNA was done using two colorectal cell lines, SW48 and HT29. These two lines were chosen because of their expression profiles for SETMAR isoforms, HT29 only expressing the V2 isoform whereas the isoforms V1, V2, X2, V5 and HSMAR1 are present in SW48.Human colorectal cancer cell lineages (SW48 and HT29) 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-HSMAR1 polyclonal sera were produced by DNA vaccination using ICANtibodiesTM technology (In Cell Art, Nantes, France) as described [doi: 10.1007/s00438-013-0754-8]. Polyclonal antibodies (pA) contained in pre-immune and anti-HSMAR1 murine sera were purified using Protein A/G MagBeads (GenScript, Piscataway, USA). Their quality was then verified by PAGE after staining with coomasie blue and their concentration was defined using the BCA Protein quantification Kit (interchim, Monluçon, France).Chromatin samples prepared from non-synchronous and exponentially growing SW48 and HT29 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-HSMAR1 pA.