GSM1530030: MCF7_40HT; Homo sapiens; RNA-Seq
Total RNA was isolated using the TRIzol Reagent (Invitrogen) from MCF-7 parental and TMR cells treated with or without 4-hydroxytamoxifen for 24 hours. RNA-Sequencing (RNA-Seq) libraries were prepared from 1 μg total RNA using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Briefly, poly-adenylated mRNA was purified from total RNA and rRNA removed by two rounds of binding to magnetic poly-T beads.  This was followed by RNA fragmentation by incubation in the presence of divalent cations at 94ºC for 5 minutes.  Double-stranded cDNA was generated by random-primed first-strand synthesis with SuperScript II reverse transcriptase and subsequent second strand synthesis with RNase H and DNA Polymerase I.  The cDNA was then blunt-ended with T4 and Klenow DNA polymerases to remove 3′-overhangs and fill in 5′-overhangs, phosphorylated with T4 PNK, and then 3′-A tailed by incubation with Klenow Fragment (3´→5´ exo–) and dATP.  Illumina paired-end (PE) adapters were ligated, followed by purification wit AMPure XP beads.  The library was enriched by high-fidelity PCR amplification (15 cycles) with Phusion DNA Polymerase (Finnzymes Oy) and adapter-specific primers.
GEO Accession
GSM1530030