Easteptm total rna extraction kit

The mRNA from cell lines was extracted using the EastepTM Total RNA Extraction Kit (Promega, Beijing, China) and reverse transcribed using the GoScript™ Reverse transcription system (Promega, Madison, USA) according to the manufacturers’ instruction. Amplification reactions were performed in ABI 7500 real-time PCR system (ABI, America), and PCR was performed using GoTaqTM qPCR Master Mix according to the manufacturers’ instruction. The Real-Time PCR primers for DAPK1, UPK2, TRAK1, ACOX1 and Glyceraldehyde-3-phosphate dehydrogenase are listed on S2 Table. PCR reaction conditions were as follows: 95°C for 2 min, 40 cycles at 95°C for 15 s and 60°C for 1 min. The cDNA from cells were used as positive control and standard curve.

For qRT-PCR, the Fgrab1DN, Fgrab1CA and PH-1 strains were inoculated in liquid CM medium and incubated at 28 °C for 16 h. Total RNA was isolated from mycelia using Eastep^TM Total RNA extraction Kit (Promega, Beijing, China), and first-strand cDNA was synthesized using M-MLV (Moloney murine leukemia virus) reverse transcriptase (HiScript III RT SuperMix for qPCR (+gDNA wiper), Vazyme, Nanjing, China). Relative transcription levels were quantified using QuantiTect SYBRgreen PCR Master Mix (Takara, Kusatsu City, Japan), using the primer pairs (Supplementary Table S2). The tubulin beta chain gene (FGSG_09530) was used as the endogenous reference gene, and the data of relative quantification were calculated using the 2^−ΔΔCT method [65 (link)]. All experiments and qRT-PCR assays were repeated three times.

The total RNAs of HepG2 cells and the excised liver were extracted with the Eastep TM total RNA extraction kit (Promega, China). The purity and concentration of RNA were determined using a micro nucleic acid protein detector (Gene 2000, Thermo) and samples with A260/A280 values ranging from 1.9 to 2.1 were selected. Reverse transcription was performed according to the instructions of Prime Script TM Synthesis Kit (Takara, China) to obtain cDNA, and the real-time fluorescent quantitative PCR reaction was carried out in the ABI 7900 H sequence detection PCR system according to the SYBR Green PCR Master Mix (Takara, China) kit instructions. The sequence of primers in the experiments was listed in Table 2.

The strains treated with TN were cultured to log-phage. Total RNA was extracted with Eastep^TM Total RNA Extraction Kit (Promega, Madison, WI, United States) and transcribed reversely to cDNA. The HAC1 splicing assay was detected by the PCR method with primers HAC1-5RT and HAC1-3RT. The PCR product was separated in agarose gel electrophoresis for 3 h (Li J. et al., 2018 (link)).