Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq)

ChIP experiments were performed as described previously [34]. Briefly, 1.6 × 10⁶ Hep3B cells were co-transfected with 6 000 ng of HIF-3α2-V5 and 4000 ng of HIF-β plasmids, cultured under normoxic conditions for 24 h and continued 24 h at 1% oxygen prior to ChIP. For the qPCR based ChIP studies, the control samples were co-transfected with 10,000 ng of pcDNA3.1-V5-HisA. Cells were crosslinked with 1% (v/v) formaldehyde and harvested for sonication to an average fragment size of 200–400 bp using Bioruptor UCD-300-TO (Diagenode, USA). The chromatin was immunoprecipitated for ChIP-seq with V5-tag antibody (R960-25, Invitrogen, USA) and for qPCR-based ChIP assays with the HIF3A antibody described above, and normal rabbit IgG (sc-2027, Santa Cruz Biotechnology, USA). ChIP-seq samples were processed according to Illumina’s instructions and DNA libraries were sequenced using Illumina HiSeq System (Illumina, USA) in the EMBL Gene Core Facility (Heidelberg, Germany). The qPCR-based ChIP results were normalized with respect to input. Fold changes were calculated using the formula 2^−(ΔCt), where ΔCt is Ct_{(immunoprecipitated DNA)} − Ct_(input) and Ct is the cycle at which the threshold line is crossed. The primers are listed in Table 1. The ChIP-seq data have been deposited in the Gene Expression Omnibus database with accession number {"type":"entrez-geo","attrs":{"text":"GSE129491","term_id":"129491"}}GSE129491.