library_construction_protocol: Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure.  RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Non strand specific library Messenger RNAwaspurified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Strand specific library Messenger RNAwaspurified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Clustering and sequencing After library quality control, different libraries were pooled based on the effective concentration and targeted data amount, then subjected to Illumina sequencing. The basic principle of sequencing is "Sequencing by Synthesis", where fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends its complementary strand, the addition of each fluorescently labeled dNTP releases a corresponding fluorescence signal. The sequencer captures these fluorescence signals and converts them into sequencing peaks through computer software, thereby obtaining the sequence information of the target fragment.