HeliScope CAGE protocol workflow. (A) Reverse transcription. cDNA is synthesized using SuperScript III and random N15 primer. (B) Oxidation/biotinylation. The cap structure is oxidized with sodium peroxide and biotinylated with biotin (long arm) hydrazide. (C) RNase I digestion. Single-strand RNA is digested with RNase I. (D) Capture on magnetic streptavidin beads. Biotinylated RNA/cDNA hybrid molecules are captured using magnetic streptavidin beads. (E) Wash unbound molecules. Unbound RNA/DNA hybrid molecules are washed away. (F) Release ss-cDNA. Captured RNA/DNA hybrid molecules are treated with RNase H and RNase I, then heat-treated. (G) Poly(A) tailing/blocking. Released cDNA is poly(A)-tailed using terminal deoxynucleotidyl transferase and dATP, then blocked with biotin-ddATP. (H) Load on flow cell. Blocked poly(A)-tailed cDNA is loaded on the HeliScope flow cell channel and anneals with the dT 50 surface. (I) Fill with dTTP/locked with A/G/C virtual terminator. After annealing of cDNA, the single-strand poly(A) tail part is filled with DNA polymerase, dTTP, and an A/G/C virtual terminator that is used in HeliScope sequencing to lock the poly(T) termini. The library is then ready for sequencing.
