RNA-Seq of citrus by-productsCitrus grandis (L.) Osbeck
Use magnetic beads with poly-T oligonucleotides to purify mRNA from total RNA. Under elevated temperature conditions, use divalent cations to fragment the mRNA into short pieces. First strand: Synthesize using random primers and M-MuLV reverse transcriptase. Second strand: Synthesize using DNA Polymerase I and RNase H. Blunt the ends of the DNA fragments through exonuclease/polymerase activity, and add an adenine deoxynucleotide (A) to their 3' ends. Ligate NEBNext adapters with hairpin loops to both ends of the fragments. Use AMPure XP beads to purify and select cDNA fragments approximately 240 bp in length. Before PCR amplification, add USER enzyme to cleave the uracil (U) in the adapters, releasing the hairpin structure. Perform PCR amplification using high-fidelity DNA polymerase and universal/index primers to enrich the library. Purify the PCR products again using AMPure XP beads, and assess the library quality using the Agilent 2100 Bioanalyzer.