Hiscript 2 1st strand cdna synthesis kit gdna wiper

Tissue samples (0.1 g) were mixed with 1 mL of PBS, ground with a mortar and freeze-thaw three times. Mouth swabs were infiltrated with 1 mL of PBS, and the suspension was centrifuged at 1000 × g for 5 min at 4 °C to obtain the supernatant. Nucleic acid extraction from the blood, tissue, and mouth swabs was performed using the Axyprep Body Fluid Viral DNA/RNA Miniprep kit (Axygen, HangZhou, China). The extracted nucleic acid was obtained using the reverse transcription HiScript II 1st Strand cDNA Synthesis kit (+ gDNA wiper) (Vazyme, Nanjing, China) to obtain cDNA, which was stored at−80 °C for further use.

Total RNA was extracted from cells using TRIzol (ET111; TransGen Biotech) according to the manufacturer’s instructions. A HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech) was used to generate the cDNA template. qRT-PCR was performed with TransStart Tip Green qPCR SuperMix (TransGen Biotech) on a LightCycler 480 II system (Roche, Basel, Switzerland) (Zhao et al., 2021 (link)). Oligonucleotide sequences (Sangon Biotech) are described in Table S2.

Total RNA from the RAW264.7 cells was isolated using the TRIzol Reagent (Invitrogen, Inc., CA, USA) and then reverse-transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme Biotech) according to the manufacturer’s instructions. qRT-PCR was performed using the EvaGreen qPCR Mastermix Kit (Vazyme AceQ® Universal SYBR® qPCR Master Mix) and CFX96 ™ real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, USA). The relative levels of the target genes were calculated using the 2^−△△Ct method, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The data of qRT-PCR was analyzed using an unpaired t-test in the GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com). For all analyses, P < 0.05 was considered statistically significant. Primer sequences are presented in Additional file 2: Table S17.

The UeEgl1 sequence was firstly predicted, by blasting egl1 of U. maydis to U. esculenta whole-genome shotgun sequencing (JTLW00000000.1). Then related primers were designed based on the predicted sequence. All the genomic DNA (gDNA) in this research was extracted by the cetyltrimethylammonium bromide (CTAB) method. Total RNA was extracted and purified using a Spin Column Fungal Total RNA Purification Kit (B518659, Sangon Biotech, China), and reverse-transcribed into complementary DNA (cDNA) with HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R212-01/02, Vazyme, Nanjing). The genomic sequence and cDNA of UeEgl1 were amplified by primers egl1-gF/gR and egl1-cF/cR separately (Table S1). Target sequences were verified by agarose gel electrophoresis and sequencing.

Plant tissues/organs were frozen in liquid nitrogen and ground into a fine powder using a mortar. Total RNA was extracted using an RNAprep pure plant kit (TIANGEN Biotech, Beijing, China) according to the instructions of the manufacturer. Then, 4 μg of total RNA of each sample was reverse transcribed using the HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China) to produce cDNA. For qPCR analysis, gene-specific primers were designed using PrimerExpress v3.0 (Applied Biosystems, Foster City, CA, USA). The qPCR assay was carried out using PowerUp^TM SYBR^TM Green Master Mix (Applied Biosystems, Waltham, MA, United States) according to the instructions of the manufacturer on StepOnePlus^TM Real-time PCR instrument (Applied Biosystems, Waltham, MA, United States). The BnActin gene was used as an internal control. Three technical replicates were conducted for each sample. The reaction procedure was as follows: 50°C for 2 min and 95°C for 5 min; followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s. Relative expression of BnMYB-related genes was calculated as described previously (Livak and Schmittgen, 2001 (link)). The primers used for PCR are listed in Supplementary Table 1.