Fastking reverse transcription kit

Only biopsies with at least 70% tumour cells were collected, and ~ 30-μm sections were cut from the FFPE samples. The Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher, Waltham, MA, USA) was used to isolate the FFPE tissue total RNA. The NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to quantify the extracted RNA. Meanwhile, the extracted RNA with an A260/A280 ratio between 1.8 and 2.2 were chosen for the quantitative RT-PCR (qRT-PCR) analysis. We used 200 ng RNA of a 20-μL reaction to reverse transcription through the FastKing Reverse Transcription Kit (Tiangen Biotech, Beijing, China). Next, we used 1 μL cDNA for PCR reaction with quantiNova PCR Kits (Qiagen, Dusseldorf, Germany) using 7900HT Fast Real-Time PCR system (Applied Biosystems, Carlsbad, USA; Indianapolis, IN). The qRT-PCR analysis was performed on all validation and independent cohort samples. The 2^−ΔΔCt method was used to calculate the expressions of interested m⁶A regulators. The details of the target m⁶A regulators primer sequences for qRT-PCR are shown in Additional file 1: Table S1.

Only the pretreatment biopsies with at least 80% of tumor cells were enrolled, and 40 μm sections were cut from pretreatment FFPE samples for RNA isolation. RNA was extracted using the Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. RNA quality and quantity were measured by NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then, 200 ng RNA was used for reverse transcription for 20 μL of reaction, using the FastKing Reverse Transcription Kit (Tiangen Biotech, Beijing, China). Finally, a total of 1 μL cDNA was used for a 10 μL PCR reaction with SYBR in the 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, IN, USA). The analysis of relative immune-related genes expression was calculated using the 2^−ΔΔCt method. Details of the commercially available mRNA primers used for qPCR are included in Supplementary Data Table S2.

For the validation of the transcriptome data, we randomly selected 6 DEGs for RT-qPCR (PCR quantitative real-time) analysis. Supplementary File 1: Table 16 lists the genes and corresponding primers used in RT-qPCR. The total RNA used in the sequencing was reversely transcribed to obtain cDNA, which was used as a template to amplify the target genes, and the RT-qPCR experiment was conducted. The RNA was reversely transcribed into cDNA using the FastKing reverse transcription kit (Tiangen, China) as per the manual’s instructions. qPCR was performed using the BIO-RAD CFX96 Real-Time qPCR system. The relative expression of each gene was calculated using 2^−△△Ct (Livak and Schmittgen, 2001 (link)).

Bacteria, cell, and tissue RNA were extracted using the total cell or tissue RNA extraction kit from Tiangen (China) and stored at −80°C, and RNA sample reverse transcription into cDNA used a FastKing reverse transcription kit from Tiangen (China). All operations were carried out according to the manufacturer's instruction. Q-PCR reaction was carried out with 1 μg of cDNA, 200 nM primer, and 5 μl of Bio-rad SYBR enzyme (USA) in a final volume of 10 μl by Bio-rad CFX96 machine. All primer sequences are available in Supplementary Table 1.

PDA plates with FOC plugs were first incubated at 28°C for 5 d, and the sublethal concentrations (2M4V and 34D: 1 μL/plate, β-C: 40 μL/plate) of different VOCs were then added separately onto the covers. After 12 h, the VOC-treated and untreated mycelia were removed, lyophilized, and ground in liquid nitrogen. Total RNA was extracted from the mycelia using a RNAsimple Total RNA Kit [Tiangen Biotech (Beijing) Co., Ltd] according to the manufacturer’s instructions. First-strand cDNA was synthesized using a FastKing Reverse Transcription Kit (Tiangen) with random hexamer primers. The resulting cDNA was used as the template for subsequent polymerase chain reaction (PCR) amplification. Quantitative real-time PCR (qRT-PCR) was performed using a Talent qPCR PreMix (SYBR Green; Tiangen) in a 7500 Fast Real-Time PCR Detection System. The actin gene was used as the internal reference for normalization. Primers for these genes (Actin, FOIG_00580, FOIG_06735, FOIG_06738, FUB2 and FUB5) are listed in S4 Table.