HIF-1a ChIPseq of mus musculus: Ba/F3 cells in 20% O2
5x107 BaF3 hMPL hJAK2 WT or BaF3 hMPL hJAK2 VF cells were incubated at the indicated O2 concentrations for 6 hours. Cells were fixed by addition of 2mM Disuccinimidyl glutarate (DSG) (Chem Cruz - Santa Cruz) for 20 minutes with agitation, pelleted by centrifugation at 500xg for 5 minutes RT, and then resuspended into 1% formaldehyde (Cell Signalling Technology) for 10 minutes at room temperature (RT), then neutralised by the addition of 10x Glycine for 5 minutes at RT. Sample processing and immunoprecipitations were performed as described previously [41,42]. Chromatin-Immunoprecipitation: Samples were then processed using SimpleChIP Enzymatic Chromatin IP Kit, Magnetic Beads (Cell Signaling Technology) according to the manufacturer's protocol. ChIP was performed with HIF-1 (D1S7W) XP Rabbit mAb (Cell Signaling Technology). DNA was isolated using a ChIP DNA Clean & Concentrator (Zymo Research). Next Generation Sequencing: Library preparation was performed using the NEBNext DNA Ultra II library preparation kit for Illumina, and performing 12 cycles PCR amplification with unique dual indexed primers. Libraries were pooled at equimolar ratios, and subject to 150 base paired end sequencing on an Illumina NovaSeq 6000 at Azenta Life Sciences.