Figure 4.

Trimming of overhanging dsRNA and adapter ligation. (A) Illustration of heterogeneous overhanging termini and their removal from dsRNA using S1 nuclease. (B) Native PAGE analysis showed that S1 nuclease cleaves overhangs but not the duplex in a range of enzyme concentrations. (C) Native PAGE analysis indicated that blunt ends generated by S1 nuclease were ligatable to dsRNA adaptors. It is notable that in the third lane from left, where only T4 DNA ligase was omitted, a faint band is observed. We cannot identify this band, which was not observed when using agarose gels. (D) RT–PCR amplification of the overhanging dsRNA that was spiked into E. coli total RNA (0.05 µg/µl) and ligated to Y-adaptors (expected PCR product length is 63 bp).