286_1_S25_mutant
The library preparation for RNA sequencing was carried out using the Illumina Stranded mRNA Prep Kit with Ligation (catalog number 20040532) as per the manufacturer's guidelines. The RNA quality and concentration were assessed using the Agilent Bioanalyzer and Qubit. Initially, total RNA input of 500 ng was subjected to poly-A mRNA enrichment using oligo-dT beads, followed by mRNA fragmentation using elevated temperature. The fragmented mRNA was then reverse transcribed into first-strand cDNA using random primers, followed by second-strand cDNA synthesis, incorporating dUTP instead of dTTP to mark the second strand. The double-stranded cDNA was purified using AMPure XP beads to remove reaction contaminants. The double-stranded cDNA underwent end repair to blunt the ends and then had an 'A' base added to the 3' ends to facilitate adapter ligation. Adapters containing unique index sequences were ligated to the cDNA fragments, allowing for multiplexed sequencing. Following adapter insertion, PCR amplification was performed to enrich the adapter-ligated DNA fragments, and the amplified libraries were purified using AMPure XP beads to remove short fragments and residual primers. Quality control checks were performed using the Agilent Bioanalyzer and Qubit to assess library size distribution and concentration. Finally, the constructed libraries were normalized, pooled in equimolar concentrations, and sequenced on an Illumina NextSeq 1000 sequencing platform according to the manufacturer's instructions.