GSM9307529: RNA-seq in T47D cells, Fulvestrant treatment, Time point 4h, replicate 3; Homo sapiens; RNA-Seq
Total RNA samples were processed using the Universal Plus mRNA-Seq with NuQuant kit (Tecan) on a Biomek i7 automated liquid handling platform (Beckman Coulter). The workflow enables the capture of polyadenylated RNA, conversion into double-stranded cDNA, and generation of strand-specific sequencing libraries. RNA quality was assessed using a Fragment Analyzer (Agilent) with the Total RNA 15 nt kit (DNF-471). Samples with RIN > 7 and DV200 > 70% were used for library preparation. A total of 250 ng of RNA in a volume of 50 µL was used as input per reaction. Library preparation was executed in two automated programs on the Biomek i7: one covering RNA purification, fragmentation, and cDNA synthesis, and a second for library construction. All steps, including poly(A) selection, RNA fragmentation, first- and second-strand cDNA synthesis, end repair, adaptor ligation with unique dual indices, strand selection, and PCR amplification, were performed according to the manufacturer's instructions using Biomek-specific labware and consumables. Reaction cleanups were performed with magnetic beads (CleanNGS or AMPure XP). Final libraries were analyzed for fragment size distribution using a Fragment Analyzer with the High Sensitivity NGS Fragment Kit (DNF-474). Library concentration was determined using the NuQuant system (Tecan) and used for equimolar pooling prior to sequencing on an Illumina NovaSeq 6000 platform. Sequencing was performed in single-end mode, generating more than 20 million reads per sample.