T1_3
Total RNA was extracted from the samples following the instructions provided with the TRlzol reagent. Genomic DNA was removed using DNaseI. The quality of RNA was assessed using a 2100 Bioanalyzer, and its concentration and purity were determined using a NanoDrop 2000. High-quality RNA samples were then utilized for sequencing library construction.RNA-specific libraries were reconstructed using Illumina TruSeq RNA Sample Preparation Kit, employing 5 g of total RNA per set. The RiboZero rRNA Removal Kit was used to eliminate rRNA, and mRNA was fragmented using Fragmentation buffer. Subsequently, cDNA reverse transcription synthesis, end-pairing, A-base separation, and junction ligation were performed based on the Illumina manual. cDNA fragments between 200 bp and 300 bp were selected through electrophoresis on a 2% agarose gel. These fragments were then amplified using a high-fidelity DNA polymerase for 15 PCR cycles. Prior to sequencing, the quality of the libraries was assessed using a NanoDrop microspectrophotometer and Labchip bioanalyzer. Finally, the paired libraries (150 bp  2) were sequenced using an Illumina NovaSeq 6000 sequencer.