RNA-Seq of Eustoma grandiflorum: EgMIXTA1 - overexpression lines 3
1. RNA sample detection The purity, concentration, and integrity of RNA samples were detected using the Nanodrop, Qubit 2.0, and Aglient 2100.2. RNA library constructionAfter the samples are qualified, the library is constructed. The main process is as follows:(1) Enrich eukaryotic mRNA with magnetic beads with Oligo (dT);(2) Add Fragmentation Buffer to randomly interrupt mRNA;(3) Using mRNA as a template, synthesize the first cDNA strand with six base random primers (random hexamers), then add buffer, dNTPs, RNase H and DNA polymerase I to synthesize the second cDNA strand, and purify it with AMPure XP beads cDNA;(4) The purified double-stranded cDNA is then repaired, A-tailed and connected to the sequencing adapter, and then AMPure XP beads are used for fragment size selection;(5) Finally, the cDNA library is obtained by PCR enrichment.3. Library quality controlAfter the library is constructed, Qubit2.0 and Agilent 2100 are used to detect the concentration and insert size of the library, and the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library.4. On-machine sequencingAfter the library is qualified, HiSeq is used for high-throughput sequencing.