title: GSM9054351: NA_IgG; Homo sapiens; OTHER
library_construction_protocol: Genome-wide binding sites of HIF1α were determined in U87 derived NA, AA, Ctrl an DMOG treated cells using the CUT&RUN Assay kit (Active motif, #53180, version 47) following the manufacturer's instructions. A total of 5x10^5 cells per line and per CUT&RUN reaction were collected and mildly fixed with 0.1% formaldehyde for 2 minutes at room temperature on a shaker. Crosslinking was quenched with 125 mM glycine for 5 minutes, and samples were washed in cold 1xPBS and flash frozen. Upon thawing, 5,000 Drosophila melanogaster nuclei (Active Motif, #53183) were added as spike-in before sample nuclei isolation.  Nuclei isolated per CUT&RUN reaction was first incubated with the concavalin beads, followed by overnight incubation with 1ug of HIF1⍺ antibody (GeneTex, GTX127309) at 4°C. Thereafter, chromatin-bound beads were mixed with pAG-MNase in cell permeabilization buffer, and the enzyme was activated by adding 1ul of 0.1M cold calcium chloride and incubated at 4°C for 2 hours while rotating at a speed of 25 RPM. Decrosslinking was performed by incubation with Stop Solution containing RNase and Glycogen at 37°C for 10min.  Enriched DNA was purified using the DNA purification Columns SF,  provided in the kit Library preparation was done using the NEBNext Ultra II DNA library Prep Kit, For Illumina and Multiplex Oligos (New England Biolabs, #E7645S, E7500S, E7335S) following Active Motif's CUT&RUN library preparation protocol. Library size distribution was assessed via TapeStation High Sensitivity DNA Analysis assay (Agilent High Sensitivity DNA screentape analysis # 5067-5592) and libraries were sequenced as PE150 on a NovaSeqX Sequencing System (Illumina).