RNA-seq of Tuber panzhihuanense/Pinus massoniana mychorrhizae
RNA integrity and the total amount have been assessed with the use of RNA Nano 6000 Assay Kit of Bioanalyzer 2100 system (Agilent technologies, CA, USA). mRNA has been isolated from total RNA with oligo(dT)-attached magnetic beads. Divalent cations were used under elevated temperature in Synthesis Reaction Buffer 5X for fragmentation. The first strand of cDNA has been synthesized through random hexamer primer and M-MuLV Reverse Transcriptase, followed by a second strand cDNA synthesis. Exonuclease/polymerase activities were applied to convert residual overhangs into blunt ends. As hybridization preparation, adaptors with hairpin loop structures were ligated following adenylation of the 3 ends of DNA fragments. cDNA fragments, preferably between 370 and 420 bp in length, were selected through library fragment purification using the AMPure XP system (Beckman Coulter, Beverly, USA). Subsequent PCR amplification was performed, and the PCR products were purified with AMPure XP beads, resulting in the final library.