Genome-Wide Identification of the FRS12 Binding Sites by Tandem Chromatin Affinity Purification - Seq Analysis in Arabidopsis thaliana

To identify the genomic binding sites of FRS12, Tandem Chromatin Affinity Purification - Seq (TChAP-Seq) was performed on 7-d-old Pro35S:FRS12-HBH and Pro35S:NLS-GFP-HBH expressing Arabidopsis thaliana PSB-D cells. Cultures were transferred to long days (16:8) conditions two weeks before harvesting at day time (DT) at zeitgeber time (ZT) 4 (time of lights on usually defines zeitgeber time zero) or night time (NT) at ZT20. Chromatin was isolated from formaldehyde-treated cell cultures following two affinity purification steps; first by IMAC using a Ni-NTA Superflow resin (Qiagen), then by a Biotin binding step using a Streptavidin Sepharose resin (GE Healthcare). Finally, protein-DNA bound fragments were decrosslinked, deproteinized and purified using QIAquick PCR Purification Kit (Qiagen). DT and NT Pro35S:FRS12-HBH samples were carried in two replicates whereas background DT and NT Pro35S:NLS-GFP-HBH samples were prepared in single replicates. The TChAP DNA samples were processed by first preparing a TruSeq ChIPseq library (Illumina) and then sequenced using Illumina HiSeq 2000 at 50bp single read at an average depth of 15 million reads.