GSM1609432: GM12891_Rep3_PolyARNA; Homo sapiens; RNA-Seq
For RNA sequencing experiments, Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Lifetechnologies), then purified using the Qiagen RNeasy kit (Qiagen, Valencia, CA) and treated with RNAse-free DNase (Qiagen, Valencia, CA). RNA integrity was checked with a Bioanalyzer (Agilent, Santa Clara CA) and only samples with an RNA integrity number (RIN) of > 9.5 were subsequently subjected to poly-A-selection. For poly-A selection, 10 μg of purified total RNA were enriched by performing two cycles of selection using the Dynabeads mRNA Purification Kit (Life Technologies). For RNA sequencing experiments, stranded libraries were prepared following the dUTP protocol (Parkhomchuk et al., 2009). In brief: ~100 ng of poly-A-selected RNA were fragmented with 10 x fragmentation buffer (Life Technologies, #AM8740) for 2 min at 70 °C. First-strand cDNA synthesis was primed with random hexamers. Actinomycin D was added to reduce anti-sense artifacts. For the second-strand synthesis dTTP was replaced with dUTP. The cDNA was end-repaired and phosphorylated with the End-It kit from Epicentre (ER0720). After treatment with Klenow fragment (NEB, Cat# M0212s) and dATP, Illumina TruSeq adapters were ligated to the protruding 3_-‘A’ base (LigaFast, Promega Cat#M8221). After size-selection on an agarose gel the second strand was eliminated by digestion with AmpErase Uracil N-glycosylase (UNG) (Life Technologies, N8080096).
GEO Accession
GSM1609432