GSM9652701: Parental MCF-7 cells_biol rep1; Homo sapiens; RNA-Seq
Total RNA was extracted from cancer cells using the Monarch Total RNA Miniprep Kit (New England Biolabs). Ribosomal RNA (rRNA) was depleted using the RiboCop rRNA Depletion Kit (Lexogen). The rRNA-depleted RNA was then used for library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer's instructions. Briefly the key stages of this protocol are successively, the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1μg total RNA (with the Magnetic mRNA Isolation Kit from NEB), a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing was performed on an AVITI platform (Element Biosciences), using 76-bp paired-end reads. Libraries were multiplexed to achieve a sequencing depth of approximately 20 million reads per sample.