Global Profiling of Polyadenylation sites Reveals Link Between Selective Polyadenylation and Cancer Metastasis
Total RNA was extract with TRIzol Reagent and treated with TURBO DNA-free™ Kit to remove genomic contamination. 50ug total RNA was incubated with streptavidin dynabeads coated with biotinylated GsuI-oligo (dT) primer. The first strand cDNA was synthesized with SuperScript III and dNTP mix containing 5-methylated-dCTP instead of dCTP. After dsDNA synthesis, DNA molecules were fragmentated to approximately 200~500 bp with fragmentase. 3' terminal fragments were released from beads through Gsu I digestion, which were subjected to deep sequencing by Illumina HiSeq 2000.