It is suggested to do the transcriptome sequencing to discover the large-scale transcripts, build the unigene library of the target species. Based on that, small RNA sequencing project can obtain the known microRNA through annotation and predict the new miRNA. The target gene of miRNA can be predicted by comparing with the gene in unigene library, and finally complete the functional annotation of the target genes.

RNA purification methods include organic solvent extraction + ethanol precipitation, silica gel membrane centrifugation column and so on. Because the silica gel membrane column can only enrich more than 200nt RNA molecules, it is not suitable for the separation and purification of small RNA. Although small RNA can be well preserved by organic solvent extraction, the later stage of precipitation is very complicated. Therefore, if small RNA sequencing is to be carried out, it is recommended not to use the column kit or the LiCl precipitation method when extracting total RNA to avoid the loss of small RNA fragments.

 The main advantages of high-throughput sequencing of small RNA includes (1) small RNA molecules can be studied directly from the nucleotide level. There is no cross reaction and background noise caused by fluorescence analog signals of traditional chip hybridization, which is very conducive to distinguish different small RNA molecules with the same family and very similar sequence; (2) It can perform high-throughput analysis on any species without any prior sequence information and secondary structure information; (3) it has high sensitivity and high through-put, which provides great data depth and coverage for the discovery and research of small RNA molecules, and can detect rare transcripts with extremely low abundance; (4) the original data generated by sequencing can be compatible with a variety of analysis software, and can annotate small RNA and its expression level. 

 Small RNA sequencing can be used to study all known small RNAs in a specific tissue of a species in a specific state. It can also find new or unique small RNAs of the species and predict their target genes, which provides a powerful tool for studying the function of small RNA and the gene regulatory mechanism of this species. Small RNA mainly includes microRNA (miRNA), small interfering RNA (siRNA), and RNA interacting with piwi (piRNA).