A consensus hypoxia signature in breast cancer

We exposed a panel of 32 breast cancer cell lines or normal human mammary epithelial cells to 20% or 1% O2 concentration for 24h. Total RNA was extracted from cells using TRIzol (Invitrogen) and treated with DNase I (Ambion). All samples had a RIN value of >9.0 when measured on an Agilent Bioanalyzer. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, end repair to generate blunt ends, A-tailing, adaptor ligation and 12 cycles of PCR amplification. Unique adaptors were used for each sample in order to multiplex samples into several lanes. Sequencing was performed on Illumina Hiseq 3000/4000 with a 150bp pair-end run. A data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. Overall design: 32 breast cancer cell lines exposedto standard tissue culture conditions normoxic (20% O2) or hypoxic (1% O2) conditions.