mRNA-Seq of THP-1 cell: human periodontal ligament stem cell-derived apoptotic extracellular vesicle treated
Following successful extraction and quality assessment of total RNA, eukaryotic mRNA is enriched using magnetic beads coated with Oligo(dT). For prokaryotic samples, ribosomal RNA (rRNA) is removed using a dedicated rRNA depletion kit. Fragmentation buffer is added to the purified mRNA, and the mixture is incubated in a thermomixer at an optimized temperature to fragment mRNA into short segments. First-Strand cDNA Synthesis: Fragmented mRNA is used as a template for first-strand cDNA synthesis. Second-Strand cDNA Synthesis: A reaction system is prepared to synthesize the second-strand cDNA using the first-strand product as a template. Purification: The double-stranded cDNA is purified using a commercial kit. End Repair and Adenylation: Blunt ends are generated via end repair, followed by adenylation of the 3' ends to enable adapter ligation. Adapter Ligation: Sequencing adapters are ligated to the adenylated cDNA fragments. DNA fragments of the desired size range are selected using a size-selection protocol. PCR amplification is performed to enrich adapter-ligated fragments and finalize the sequencing library. The constructed library undergoes rigorous quality assessment using an Agilent 2100 Bioanalyzer and Qubit fluorometer. Qualified libraries are subjected to high-throughput sequencing on an Illumina platform.