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Total RNA was extracted by Trizol. Totally, 0.1% agarose gel electrophoresis was performed to determine the RNA integrity and eliminate DNA contamination. RNA purity (OD260/280 and OD260/230) was determined by a Nanophotometer Spectrometer and RNA concentration was quantified by a QUBIT2.0. Library construction was performed under the guidance of a Small RNA Sample Pre Kit . Briefly, the 3 SR and 5 SR adaptor were ligated and the first chain was subsequently synthesized by reverse transcription. After PCR amplification, target DNA fragments were separated by PAGE electrophoresis and cDNA library was obtained after gel extraction. At last, PCR products were purified and the quality of sequenced samples was assessed by calculating probabilities of error for each base-call using Phred .The clustering of the index-coded samples was conducted on a cBot Cluster Generation System with TruSeq PE Cluster Kit v4-cBot-HS (Illumia) based on the manufacturers instruction. After cluster generation, the library preparations were sequenced on an Illumina platform and single-end reads were generated. Raw data of fastq format was firstly processed through in-house perl scripts. In this step, clean data was obtained by removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data. Reads shorter than 18 nt or longer than 30 nt were also trimmed. At the same time, quality score (Q-score), GC-content, and sequence duplication level of the clean data were calculated.