RNA-seq of Homo sapiens:U251-ORF-3
Total RNA was extracted from the samples, followed by quality and integrity assessment of the RNA samples using Nanodrop, Qubit, and Agilent 2100 Bioanalyzer. After the samples passed the quality control (QC), mRNA was enriched using mRNA Capture Beads, and then fragmentation of the mRNA was achieved via heat treatment in the presence of divalent metal ions. Using the mRNA as the template, first-strand cDNA was synthesized with random hexamers, followed by a second-strand cDNA synthesis reaction. The double-stranded cDNA was then purified with DNA Clean Beads. The purified double-stranded cDNA was subjected to end repair, A-tailing, and ligation of sequencing adapters, followed by size selection using DNA Clean Beads. Finally, PCR amplification was performed, and the PCR products were purified with DNA Clean Beads to obtain the final libraries. After library construction was completed, preliminary concentration quantification was performed using Qubit 3.0, followed by insert size verification of the libraries using the Agilent 2100 Bioanalyzer. Once the insert size met the expected criteria, accurate quantification of the effective library concentration was carried out using the ABI Step One Plus Real-Time PCR System. After the libraries passed the QC, sequencing was performed on each qualified library.