Illumina NovaSeq X paired end sequencing of SAMD01582613
Total RNA was extracted using the NucleoSpin RNA Kit from AT24 strain, which is a RIB40 mutant overexpressing AO090010000714, cultured for 96 hours in a starch-based CD medium. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation and first-strand cDNA synthesis were performed using random hexamer primers. Second-strand cDNA was synthesized using dUTP instead of dTTP to retain strand specificity. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, including end repair, A-tailing, adapter ligation, USER enzyme digestion, PCR amplification, and purification. Sequencing was performed on the Illumina NovaSeq X Plus platform with paired-end 150 bp reads.