Large scale profiling of piRNAs from silkworm 24 hours after fertilization embryos
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Total RNAs were prepared using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA (10 microgram) was loaded onto a 15% denaturing polyacrylamide gel containing 10 M urea, electrophoresed, and then stained with SYBRGold (Invitrogen). Signals were visualized using LAS-1000 film (Fujifilm). Small RNA libraries were constructed using a Small RNA cloning kit (Takara). DNA sequencing was performed using the Solexa genetic analysis system (Illumina) (Bently, 2006). One nano-gram of the prepared cDNA was used for the sequencing reactions with the Illumina GA. 10,000-15,000 clusters were generated per "tile" and 36 cycles of the sequencing reactions were performed. The protocols of the cluster generation and sequence reactions were according to the manufacturer's instructions.