Gdna wiper

To measure the relative expression levels of MoSNF5 at different developmental stages, whole RNAs were extracted with an RNA extraction kit (Invitrogen, Waltham, MA, USA) from different tissues, including mycelia cultured in liquid CM, conidia produced on OTA plates, appressoria formed on hydrophobic film surfaces, and infected-barley leaves inoculated with conidial suspensions. The crude RNA was pre-treated with gDNA wiper (Vazyme, Nanjing, China) and was then reversely transcribed with HiScriptR II Reverse Transcriptase (Vazyme). Quantitative RT-PCR (RT-qPCR) was performed on an ABI 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. The stable-expression actin gene (MGG_03982) was used as an internal control. For comparing the relative abundance of target transcripts, the mycelial sample was used as a calibrator and the average threshold cycle (Ct) was normalized to the actin gene as previously described [42 (link)]. When comparing the relative abundances of target genes between the wild-type strain and mutants, the wild-type sample was used as a calibrator. The experiments were repeated three times, with three replicates each time.

RNA was extracted from the early embryos as described above. cDNA was then synthesized using HiScript II Q Select RT SuperMix for qPCR with gDNA wiper (Vazyme #R233-01) according to the manufacturer’s instructions. Real-time PCR was performed on a CFX Connect Thermal Cycler (Bio-Rad) with ChamQ SYBR qPCR Master Mix (Vazyme # Q311-02). Amplification was performed with a two-step reaction at 95°C for 3 min and 40 cycles at 95°C for 15 s and at 60°C for 30 s. A 20 μL PCR mixture included 10 μL ChamQ SYBR qPCR Master Mix, 2 μL primer mix (10 μM each), 3 μL diluted template cDNA, and 5 μL deionized distilled water. The relative fold changes in related genes were normalized to expression levels of actin, and analyzed by 2-way ANOVA followed by Dunnett’s multiple comparisons. Each experiment was repeated four times. The PCR primers used in this study are listed in Supplementary Table 2.

Total RNA from different cells was isolated from PBS-washed cells using Trizol reagent (Invitrogen). Isolated RNAs were used as templates for reverse transcription based on the manual for HiScript III 1st Strand cDNA Synthesis Kit plus gDNA wiper (Vazyme Biotech). Real-time PCR analysis was conducted with ChamQ SYBR qPCR Master Mix (Vazyme Biotech) and carried on a QuantStudio three Real-Time PCR System (Applied Biosystems). Primers for amplifying each target were listed in Supplementary Table S1.

Total RNA was extracted from colon tissue or cells using the TRIzol reagent (Invitrogen). Reverse transcription was performed using HiScript® Q RT SuperMix with a gDNA wiper (Vazyme), according to the manufacturer's instructions. Real-time PCR was performed using ChamQ SYBR® Color qRT-PCR Master Mix (Q711, Vazyme). The relative expressions of lncRNA were calculated using the 2^-∆∆Ct method. Primers of lncRNAs were designed and synthesized by Sangon Biotech, China. All primers are shown in Supplementary Table 1. GAPDH was employed as an endogenous control.