RNA-Seq of Korla fragrant pears: leaves
RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (each sample was guaranteed to be approximately 100 mg), and the extraction process was carried out according to the instructions. After RNA extraction, a Nanodrop was used to determine the RNA purity (OD260/280) and concentration and to determine whether the nucleic acid absorption peak was normal. The integrity of the RNA was accurately detected with an Agilent 2100 instrument. The detection indicators included the following: the RIN value, 28S/18S ratio, whether the chromatogram baseline was increased, and the 5S peak. After the samples passed the test, they were transported on dry ice to Xinjiang Aidesen Biological Co., Ltd. (Urumqi, China) for sequencing. Magnetic beads with oligo(dT) are used to enrich mRNAs by binding to the poly(A) tail of the mRNA through A-T complementary pairing. Then, fragmentation buffer was added to break the mRNA into short fragments. Random hexamers were used to synthesize first-strand cDNA using mRNA as a template. Then, buffer, dNTPs and DNA polymerase I were added to synthesize second-strand cDNA. The double-stranded cDNA was then purified using AMPure XP beads. The purified double-stranded cDNA was then end-repaired, A-tailed and connected to sequencing adapters, the fragment size was selected using AMPure XP beads, and finally, PCR enrichment was performed to obtain the final cDNA library. The library was detected using Agilent 2100 and qPCR methods. The constructed library was sequenced on the Illumina HiSeq 2500 sequencing platform.