GSM2670759: m6_Ovaries; Homo sapiens; RNA-Seq
Total cellular RNA was isolated from snap-frozen cell pellets (73,942- 433,703 cells) using the TRIzol reagent (Life Technologies) and a modified protocol that incorporatesd an additional extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 4.3).  RNA quantity and quality were assessed on a NanoDrop spectrophotometer (Thermo Scientific) and the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. RNA-Sequencing (RNA-Seq) libraries were prepared from 1 ug 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 ribosomal RNA 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 94C for 5 minutes.  Double-stranded cDNA was then 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 the 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 then ligated, followed by purification wit AMPure XP beads.  The library was then enriched by high-fidelity PCR amplification (15 cycles) with Phusion DNA Polymerase (Finnzymes Oy) and adapter-specific primers.
GEO Accession
GSM2670759