GSM9595703: MCF-7 parental (Vehicle)-2; Homo sapiens; RNA-Seq
RNA was isolated from the cells using Trizol reagent (Thermo Fisher). RNA samples were quantified for RNA and DNA content using a Qubit fluorometer (Invitrogen) and analyzed for integrity using Agilent 4200 TapeStation. Sequencing libraries for Illumina sequencing were prepared in one batch in 96-well plate using CORALL Total RNA-seq Library Prep Kit with Unique Dual Indices with RiboCop HMRv2 rRNA Depletion Kit (Lexogen). In brief, approximately 7–80 ng of total RNA per sample were used for the first rRNA depletion step, then followed by library generation initiated with random oligonucleotide primer hybridization and reverse transcription. Next, the 3′ ends of first-strand cDNA fragments were ligated with a linker containing Illumina-compatible P5 sequences and Unique Molecular Identifiers (UMIs). During the following steps of second strand cDNA synthesis and ds cDNA amplification, i7 and i5 indices, as well as complete adapter sequences required for cluster generation, were added. The number of PCR amplification cycles was 15 as determined by qPCR using a small pre-amplification library aliquot for each individual sample. Final amplified libraries were purified, quantified, and average fragment sizes were confirmed to be approximately 320–370 bp by gel electrophoresis using 4200 TapeStation and D5000 Screen Tape (Agilent). The concentration of the final library pool was confirmed by qPCR and the pool was subjected to test sequencing on MiniSeq instrument (Illumina) in order to check sequencing efficiencies and adjust accordingly proportions of individual libraries. Sequencing was carried out on NovaSeq 6000 (Illumina), S4 flowcell, 2/150 bp, and 60 paired-end reads per sample. It was done at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.