RNA-seq of human breast cancer
Total RNA was extracted from either fresh-frozen tumor samples or formalin-fixed, paraffin-embedded (FFPE) tissue sections when fresh-frozen samples were unavailable. RNA integrity was assessed using the Agilent Tapestation 4200. RNA concentration was measured using the Invitrogen Qubit RNA HS Assay Kit (ThermoFisher Scientific). Fresh-frozen samples with an RNA Integrity Number (RIN) between 2.0 and 9.0 were included for downstream analyses, while FFPE samples had RIN values ranging between 1.5 and 2.5. Library preparation was conducted at the Science for Life Laboratory (National Genomics Infrastructure, Uppsala). Illumina TruSeq strand-specific RNA libraries were generated, incorporating ribosomal RNA depletion using the RiboZero human kit. Paired-end sequencing (125 bp) was performed on an Illumina HiSeq2000 platform, yielding approximately 15 to 30 million aligned reads per sample (median: 22.5 million aligned reads). Read alignment and computations were carried out using resources provided by the Swedish National Infrastructure for Computing (SNIC) through UPPMAX. Reads were aligned using STAR aligner and gene expression levels were quantified using RSEM. Data were normalized using variance stabilizing transformation (VST) and batch effects were corrected using the removeBatchEffect function from the limma package. A total of 11 FFPE samples were available, 10 of which had matching clinical data and were included in downstream analysis. Data quality was assessed by quality control metrics including library complexity, entropy, gene detection rate, and principal component analysis. FFPE-derived samples (batch 3) showed reduced library size and detected genes but did not form distinct clusters in the PCA space post-batch correction, Figure S1. Therefore, all samples were retained to maximize statistical power.  A total of 428 samples, of which 344 (355 after imputations, see below) were ER-positive, had available clinical and molecular information.