GSM5766329: MDA-MB-231-BRL-50481 treated replicate 3; Homo sapiens; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen) as per the instruction from the manufacture and then purified using RNAeasy mini columns (Qiagen) as per the manufactures instructions. mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.