1. DNA digest: total RNA was treated with DNase I which degraded double-stranded and single stranded DNA presenting in RNA samples.; 2. rRNA removal: using the Ribo-off rRNA Depletion Kit to remove ribosomal RNA.; 3. RNA fragmentation.; 4. cDNA Synthesis: First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP.; 5. End repair, add A and adaptor ligation.; 6. PCR amplification: The PCR reaction system and program are configured and set up to amplify the product.; 7. Library quality control.; 8. Cyclization: Denature the PCR product by heat and the DNA is cyclized by splint oligo and DNA ligase.; 9. DNA nanoball (DNB) synthesis.; 10. Sequencing on DNBSEQ platform.