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. 2014 Jul 8;9(7):e101812. doi: 10.1371/journal.pone.0101812

Figure 4. Library preparation using the Non-CapSMART method.

Figure 4

A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides (Table 2: STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.