RNA-Seq of melon stem
Total RNA ExtractionTotal RNA is extracted from tissue samples, and its concentration and purity are measured using a Nanodrop 2000. RNA integrity is checked by agarose gel electrophoresis and the RQN is determined using an Agilent 5300. For library preparation, the RNA should have a total mass of 1g, concentration  30 ng/L, RQN > 6.5, and OD260/280 between 1.8 and 2.2.Oligo dT Enrichment of mRNAEukaryotic mRNA has a polyA tail at the 3' end. Magnetic beads with Oligo(dT) sequences are used to bind the polyA tail, allowing mRNA to be separated from total RNA for transcriptome analysis.mRNA FragmentationSecond-generation sequencing platforms sequence short fragments. The enriched mRNA is fragmented into approximately 300bp pieces using a fragmentation buffer.Reverse Transcription to cDNAUsing reverse transcriptase and random primers, mRNA is reverse transcribed into single-stranded cDNA, followed by second-strand synthesis to form a stable double-stranded structure.Adaptor LigationThe double-stranded cDNA, with sticky ends, is converted to blunt ends using an End Repair Mix. An A base is added to the 3' end to facilitate adaptor ligation.Fragment Selection and Library EnrichmentAdaptor-ligated products are purified and size-selected, followed by PCR amplification to obtain the final library.SequencingNovaSeq X Plus Platform SequencingQuantify using Qubit 4.0 and mix libraries according to data proportions.Perform bridge PCR on the cBot to generate clusters.Sequence the libraries.DNBSEQ-T7 Platform SequencingCircularize the library DNA.Remove non-circularized sequences and purify.Prepare DNA nanoballs (DNB).Note: Follow the sequencing steps corresponding to the chosen high-throughput sequencing platform.