GSM2218831: MCF10A MII_siControl, TGFβ 16h_RNA; Homo sapiens; RNA-Seq
RNA was isolated using the RNeasy Kit (Qiagen). RNA-seq libraries were prepared essentially as described (Parkhomchuk et al., 2009). In short, mRNA was isolated from 1 μg total RNA using Dynabeads Oligo(dT)25 (Life Technologies) and fragmented to 150-200 nt in first strand buffer for 3 min at 94°C. Random hexamer primed first strand was generated in presence of dATP, dGTP, dCTP and dTTP. Second strand was generated using dUTP instead of dTTP to tag the second strand. Subsequent steps to generate the sequencing libraries were performed with the NEBNext kit for Illumina sequencing (New England Biolabs) with minor modifications; after indexed adapter ligation to the dsDNA fragments, the library was treated with USER (Uracil-Specific Excision Reagent) Enzyme (New England Biolabs) in order to digest the second strand derived fragments. After amplification of the libraries, samples with unique sample indexes were pooled and sequenced using HiSeq 2500 with TruSeq SBS Kit v4 reagent (Illumina) following the manufacturer's protocols.
GEO Accession
GSM2218831