design: RNA-seq analysis of human breast cancer cell lines MCF7 and MDA-MB-231 treated with imipramine compared to untreated controls
library_construction_protocol: Both cell lines were seeded in 6-well plates at a density of 100,000 cells/well per sextuplicate and treated with 20 µM of imipramine.  Cells were collected after 24, 48, and 72 h (with or without treatment) using 800 µL of TRIzol™ Reagent (Thermo Fisher Scientific). Total RNA was extracted using the Direct-zol™ RNA MiniPrep kit (Zymo Research, USA, Cat# R2050). RNA sequencing was performed at the Genomics Department of the Institute of Biomedicine in Murcia (IMIB), using the Illumina NextSeq 2000 platform. A total of 1000 ng of RNA per sample was used for library preparation using the Illumina Stranded mRNA Prep Kit following the manufacturer's instructions. Briefly, oligo(dT) magnetic beads were used to purify and capture mRNA molecules containing polyA tails. Purified mRNA was fragmented and reverse-transcribed into the first strand of complementary DNA (cDNA) using random primers. In the second-strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. In the final steps, adenine (A) and thymine (T) bases are added to the ends of the fragments and adapters are attached. The resulting products were purified and selectively amplified for sequencing using the Illumina system.