RNA-Seq of homo sapiens
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dTTP for non strand specific library or dUTP for strand specific library.For the non strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.Raw sequencing reads (FASTQ) were trimmed to remove adapters, low-quality bases, and poly-N sequences. Clean reads were mapped to the reference genome using Hisat2 v2.0.5. Gene expression levels were quantified using featureCounts v1.5.0-p3. Differential expression analysis was performed with DESeq2 or edgeR, and functional enrichment analyses (GO, KEGG, Reactome, DO, DisGeNET) were carried out using clusterProfiler.