GSM4310136: MCF-7 rep5_RNA-seq; Homo sapiens; RNA-Seq
Standard methods were used for RNA-seq library construction, EZBead preparation, and sequencing, based on the Life Technologies SOLiD 5500xl system. Briefly, 2 μg of total RNA per sample was used for library preparation. The rRNA was first depleted using the standard protocol of RiboMinus Eukaryote Kit for RNA-Seq (Ambion), and rRNA-depleted RNA was concentrated using a PureLink RNA Micro Kit (Invitrogen). Whole transcriptome libraries were prepared and individually barcoded using the the SOLiD Total RNA-seq kit (Life Technologies). Each barcoded library was quantified by quantitative polymerase chain reaction (qPCR) using SOLiD Library Taqman qPCR Module (Life Technologies) and pooled in equal molarity. EZBead preparation, bead library amplification, and bead enrichment were then conducted using the EZ Bead E80 System (Life Technologies). Finally, sequencing by ligation was performed using a standard single-read, 5'-3' strand-specific sequencing procedure (75 nt-read) on a SOLiD 5500xl instrument.