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. 2016 Dec 16;7:13783. doi: 10.1038/ncomms13783

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Copyright © 2016, The Author(s)

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Capp-Switch includes (a–c) capture of 5′ mRNA fragments and (d–f) cDNA synthesis and sequencing. (a) The mRNA 5′ triphosphate is capped with biotin-GTP by VCE. (b) RNA is fragmented and (c) the capped 5′ mRNA fragments are captured on streptavidin magnetic beads and separated from other RNA. (d) The 5′ mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV reverse transcriptase. An oligonucleotide hybridizes to the 3′ overhang and the complementary sequence is synthesized by the MMLV template-switching activity. (e) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. (f) The cDNA is sequenced on a high-throughput platform.