The day after U251s were seeded in the collagen hydrogels, media was refreshed and well plates or flasks were placed in an incubator at normoxic (21% O2) or hypoxic (1% O2) conditions. Samples were incubated for 72 hours. Cells in flasks were trypsinized, rinsed once in ice cold PBS, then re-suspended in ice cold PBS at 1,000 cell/μl. Cells seeded in collagen hydrogels were obtained by digesting the collagen in a solution of 0.5% collagenase (Thermo Fisher, Waltham, MA) and 1% FBS in Hanks Buffered Salt Solution (HBSS)( Lonza). Collagen scaffolds were submerged in collagenase solution and incubated at 37˚C and 5% CO2 for 2 hours. Digested collagen solution was collected, rinsed once in ice cold PBS, and re-suspended in ice cold PBS at 1,000 cell/μl. 1 μl of phenylmethyldulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO) and 1 μl of protease inhibitor cocktail (1X concentration) (Sigma-Aldrich, St. Louis, MO) were added to a 100 μl aliquot of cell suspension for each culture condition. All ChIP-seq libraries were constructed using Accel-NGS 2S plus DNA library kit (Swift Bioscience) following the manufacturer's instructions.