RNA-Seq of ING4 over-expressed 786-O cells
After the samples passed the assay, the rRNA was removed with the Ribo-zero kit. subsequent fragmentation of RNA was achieved using high temperature and metal ion action. Ribosomal-depleted RNA was used as a template to synthesize one-stranded cDNA with six-base random primers (random hexamers), followed by the addition of buffer, dNTPs (dUTP, dATP, dGTP and dCTP) and enzymes to synthesize two-stranded cDNA, followed by purification using VAHTSTM DNA Clean Beads. The purified double-stranded cDNA was first end-repaired, A-tailed and sequenced with sequencing junctions, and fragment size sorted using VAHTSTM DNA Clean Beads. The final strand-specific cDNA library was obtained by PCR enrichment and purification of the PCR products with VAHTSTM DNA Clean Beads. After the library was constructed, the concentration was initially determined using Qubit 3.0, followed by detection of the library insert using an Agilent 2100 Bioanalyzer, and the effective library concentration was accurately quantified using the ABI Step One Plus Real-Time PCR system.