RNA-Seq of peripheral blood mononuclear cells
Total RNA was isolated from PBMCs using TRIzol reagent through phase separation, precipitation, and washing steps. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer, and its integrity was assessed by agarose gel electrophoresis. Libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina for . Poly(A)+ mRNA was enriched using oligo(dT) magnetic beads and randomly fragmented. The fragmented mRNA was reverse-transcribed to synthesize cDNA, which was then purified and ligated with sequencing adapters. cDNA fragments (approximately 400500 bp) were size-selected, PCR-amplified, and purified to generate sequencing-ready libraries. Library quality was evaluated with an Agilent High Sensitivity DNA Kit on an Agilent 2100 Bioanalyzer to ensure quality standards.