title: APP-T2
design: Total RNA was isolated and purified using TRIzol following the manufacturer's protocol. Quality control of the total RNAquantity and puritywas assessed using a NanoDrop ND-1000 and its integrity was evaluated using a Bioanalyzer 2100. Criteria met included a concentration >50ng/L, an RIN value >7.0, and total RNA >1g suitable for downstream experiments. Poly(A) mRNA was specifically captured via two rounds of purification using oligo(dT) magnetic beads. The captured mRNA was then fragmented under high-temperature conditions using the NEBNextR Magnesium RNA Fragmentation Module at 94C for 5-7 minutes. The fragmented RNA was converted to cDNA by reverse transcriptase. Double-strand synthesis was carried out using E. coli DNA polymerase I and RNase H, converting the DNA-RNA hybrids into double-stranded DNA while incorporating dUTP Solution to blunt the ends. An 'A' base was added to each end, facilitating the attachment to adapters with terminal 'T' bases, and the fragments were size-selected and purified using magnetic beads. The double strands were digested with UDG enzyme, followed by PCR amplificationpre-denaturation at 95C for 3 minutes, total of 8 cycles of denaturation at 98C for 15 seconds, annealing at 60C for 15 seconds, and extension at 72C for 30 seconds, with a final extension at 72C for 5 minutes, producing a library with fragment sizes around 300bp  50bp (strand-specific library). Finally, we conducted paired-end sequencing using the Illumina NovaSeq 6000 in standard operation mode PE150.