Transcriptome sequencing of Ophiocodyceps sinensis at different harvest stages

The Ophiocordyceps Sinensis complete mRNA sequences of sclerotia germination stage, sclerotia development stage and mature stage of sclerotia were determined by RNA-seq. The samples were collected from Zaduo County, Yushu City, Qinghai Province, China. transcriptome sequencing based on next-generation sequencing (NGS) technology of Illumina HiSeq sequencing platform. Total RNA was extracted from each sample using Trizol reagent (Invitrogen, Carlsbad, USA) gel electrophoresis was used to evaluate RNA integrity, and a NanoDrop NC2000 ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and Agilent 2100 bioanalyzer (Beijing, China) were used to measure RNA concentration and quality. High-quality RNA samples were selected for constructing sequencing libraries. RNA purification, reverse transcription, library construction, and sequencing were performed at Majorbio bio-pharma Biotechnology Ltd (Shanghai, China) according to the manufacturer's instructions (Illumina, San Diego, CA). Poly(A) mRNA was purified from total RNA using poly- T oligo-attached magnetic beads, and it was fragmented into short fragments of 300 bp by adding fragmentation buffer. Double-stranded cDNA was synthesized using SuperScript Double-stranded cDNA Synthesis Kit (Invitrogen, CA) and random hexamer primers (Illumina). Finally, End Repair Mix was added to the synthesized cDNA for end repair, phosphorylation and 'A base' addition. The cDNA target fragment of 300 bp library was selected on 2 percent of Low Range Ultra agarose, and then 15 PCR cycles were amplified using Phusion DNA polymerase (NEB). After quantification by Qubit 4, paired-end RNA-seq sequencing libraries were sequenced using a NovaSeq.