BT549, CAL-120, and MDA-MB-468 cells were preincubated in DMEM-F12 or Plasmax (both supplemented with 2.5% FBS) for 2 days. Subsequently (day 2), cells were seeded in six-well plates at 50,000 cells per well (BT549 and CAL-120) or 100,000 cells per well (MDA-MB-468) in 2 ml of the respective culture medium. After 24 hours (day 3), culture medium was changed to 8 ml of the respective medium, and cells were further incubated for 72 hours under atmospheric (21% O2, normoxia) or hypoxic (0.1% O2, hypoxia) conditions. On day 6, cell number was determined using a Coulter counter (n = 3 wells per condition), and cells were harvested for RNA extraction [n = 1 well per condition; samples were processed using the QIAshredder/RNeasy Mini Kit (Qiagen)].

For RNA-seq, samples were processed with on-column deoxyribonuclease digestion and diluted to 1.5 μg of RNA per 50 μl of water, and the quality of the purified RNA was tested on an Agilent 2200 TapeStation using RNA screentape. Libraries for cluster generation and DNA sequencing were prepared following an adapted method from Fisher et al. (20) using Illumina TruSeq RNA Library Preparation Kit v2. Quality and quantity of the DNA libraries were assessed on an Agilent 2200 TapeStation (D1000 screentape) and Qubit (Thermo Fisher Scientific), respectively. The libraries were run on the Illumina NextSeq 500 using the High Output 75 cycles kit (2 × 36 cycles, paired-end reads, single index). Quality control of the raw RNA-seq data files was performed by FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastq_screen (www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). Then, RNA-seq reads were aligned to the human genome (GRCh38.75) using TopHat2 (21), and resulting bam files were processed with htseq_count (https://htseq.readthedocs.io/en/release_0.10.0/). The final counts were normalized and analyzed with DESeq2 (22). Statistically significant differences in gene expression were determined with an FDR of 10%. In the PCA, the first principal component explains 85.1% of the variance. The second, third, and fourth components explain 8.9, 5.8, and 0.1% of the variance, respectively.

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