mRNA-Seq of THP-1 cell: PBS 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.