title: Transcriptomic profiling of AR knockdown in human triple-negative breast cancer Hs578T cells
design: Transcriptomic profiling of AR knockdown in human triple-negative breast cancer Hs578T cells
library_construction_protocol: Hs578T parental and radioresistant (RR) cells were harvested 24 hours after transfection for RNA extraction. Cells were washed twice with cold PBS and collected directly from culture dishes using cell scrapers, ensuring consistent handling across all samples. Hs578T parental and RR cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 25 mM HEPES. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. For gene knockdown experiments, cells were transfected with control siRNA (sc-37007) or AR siRNA (sc-29204) using Lipofectamine RNAiMax (Invitrogen, 13778-150) following the manufacturer's instructions. siRNAs were diluted to a final concentration of 10 nM in Opti-MEM and incubated for 15 min with Lipofectamine RNAiMax at a 1:1 ratio before being added to the cells. Total RNA was extracted using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. RNA integrity was verified using an Agilent 2100 Bioanalyzer before library preparation. RNA-seq libraries were constructed using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Poly(A)+ mRNA was purified from total RNA, fragmented, and reverse transcribed to cDNA. After adapter ligation and PCR amplification, libraries were quantified and quality-checked prior to sequencing.