design: RNA-Seq of ERα-positive breast cancer cell lines to measure the interplay of fulvestrant treatment and hypoxic conditions
library_construction_protocol: MCF7 or T47D cells were seeded into 10 cm dishes and incubated at 37°C with 5% CO₂ for 24 hours. Cells were then treated with either 100 nM fulvestrant or ethanol vehicle control and maintained under normoxic conditions for 48 hours. Subsequently, dishes were either kept in normoxia or transferred to a hypoxic workstation for an additional 48 hours (1% Oxygen). At the end of the incubation period, cells were rinsed with PBS, trypsinised at 37°C for 5 minutes, and the trypsin was neutralised with culture medium. Cells were centrifuged at 300 × g for 5 minutes, washed once with PBS, and centrifuged again at 500 × g for 5 minutes. The resulting cell pellet was resuspended in PBS, treated with RNAlater (Sigma) for RNA preservation, and stored at −80°C until RNA extraction. Total RNA was extracted using the Monarch Total RNA Mini Kit (New England Biolabs) according to the manufacturer's instructions. RNA quality and concentration were assessed using an Agilent Bioanalyzer 2100 and Qubit fluorometer, respectively.  Illumina-compatible RNA-seq libraries were prepared by the University of York Bioscience Technology Facility (BTF) Genomics Lab. Poly(A)+ RNA was isolated, fragmented, and reverse transcribed into cDNA. Libraries were constructed through end repair, A-tailing, adapter ligation, and PCR amplification following standard Illumina protocols. 