RNA-Seq of MCF7 cells in 5% O2/Plasmax
Total RNA was extracted from MCF7 and PC-3 cells cultured under different oxygen and medium conditions using the RNeasy Mini Kit (QIAGEN). RNA quality and integrity were assessed by spectrophotometry, agarose gel electrophoresis, and Agilent 2100 analysis. Equal amounts of RNA from biological replicates were pooled per condition and sent to Novogene for library preparation. Polyadenylated RNA was enriched using oligo(dT) beads, and rRNA was removed with the Ribo-Zero kit. Fragmented RNA was reverse-transcribed to cDNA using random hexamer primers, followed by adaptor ligation, size selection, and PCR enrichment. Libraries were quality-checked by Qubit, Agilent 2100, and qPCR before sequencing on an Illumina NovaSeq 6000 S4 platform (paired-end 150 bp).