Small RNA deep sequencing of eyestalks
Small RNA libraries were constructed using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, MA, USA), following the manufacturers protocol. Briefly, 1 g of RNA was ligated to 3 and 5 adapters using Ligation Enzyme Mix, then reverse-transcribed using Superscript II reverse transcriptase. Samples were then PCR-amplified. The library was then sequenced on a HiSeq platform (Illumina) by Shanghai Personal Biotechnology. Quality control of raw reads was performed in FastQC (Babraham Bioinformatics, Cambridge, United Kingdom). Clean reads with sequence length greater than 18 nt and less than 36 nt were counted. Identical sequences in a single sample were reprocessed and sequence abundance was determined. These sequences were considered unique reads for subsequent analysis.