Total RNA was extracted using the Trizol(Invitrogen, CA, USA), RNA purity and integrity was monitored by NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and a Bioanalyzer 2100 system (Agilent  Technologies, CA, USA). RNA contamination was assessed by 1.5% agarose gel. Oligo(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA  was fragmented into small pieces with fragment buffer at appropriate temperature. Then  First-strand cDNA was generated using random hexamer-primed reverse transcription,  followed by a second-strand cDNA synthesis and purified using AMPure XP Beads.  Afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end  cDNA repair. The cDNA fragments obtained from previous steps were amplified by  PCR, and products were purified by Ampure XP Bead, get the final library. After the library was constructed, Qubit 2.0 was used for preliminary  quantification, and the library was diluted to 1.5 ng/μL. Then Agilent 2100 BioAnalyzer  was used to detect the insert size of the library. Qrt-pcr was used to accurately quantify the effective concentration of the library to ensure the quality of the library.