GSM5990332
Fungi RNA was extracted using the TRIzol® method following the manufacturer's protocol. Bulk cells or filamentous fungus are grounded to a powder using liquid nitrogen, and the powder was transferred into the 2ml tube contains 1.5ml Trizol reagent. The mix was shaked for 3min, and kept for 5min at room temperature, then was Centrifuged at 10,000×g for 5min at 4°C. The supernatant was added 200uL of chloroform/isoamyl alcohol (24:1) with 1ml lysis reagents. After centrifuged at 10,000×g for 10min at 4°C, the supernatant was transferred into another new tube with equal volume of isopropanol and put in the refrigerator at -20°C for 1h. After centrifuged at 13600×g for 20min at 4°C, the supernatant was precipitated by 1 mL of 75% ethanol and let dry for 3-5min. The RNA pellet was dissolved with 30-100uL of DEPC water or RNase-free water. The concentration of the extracted RNA samples was determined using a Nanodrop system (NanoDrop, Madison, USA), and the integrity of the RNA was examined by the RNA integrity number (RIN) using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA). DNase I was used to digest double-stranded and single-stranded DNA in total RNA, then, magnetic beads were purified to recover the reaction products, RNase H method (Illumina, USA) was used to remove the rRNA. Purified mRNA from previous steps was fragmented into small pieces with fragment buffer at appropriate temperature. Then, First-strand cDNA was generated in First Strand Reaction System by PCR, and the second-strand cDNA was generated as well. The reaction product was purified by magnetic beads, afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to carry out end repair. The cDNA fragments with adapters were amplified by PCR, and the products were purified by Ampure XP Beads. Library was validating on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products above were heated denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29(Thermo Fisher Scientific, MA, USA) to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China).