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Fig. 1 Outline of the experimental protocol used for preparation of small RNA libraries. RNA was isolated from ZF developmental samples and from adult tissues using TRIzol® and fractionated on 12.5% denaturing PAGE. Small RNAs were purified from those gels and then ligated to a 3′ adapter (AMP-5′p-5′p/CTGTAGGCACCATCAATdi-deoxyC- 3′) and to a 5′ linker (see Methods). cDNA was prepared and amplified using 20 PCR cycles. PCR products were subjected to clonal amplification by emulsion PCR and then pyrosequenced using a 454 genome sequencer.
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