Superrt cdna synthesis kit

Total RNA was extracted from A. tubingensis and A. apis using ZN Fungal/Bacterial RNA Prep (TIANMO Biotech, Beijing, China), and cDNA was synthesized using tagged specific primer (Supplementary Table S3) with super RT cDNA Synthesis Kit (CW Biotech, Beijing, China). The cDNA synthesis mixture was incubated at 42°C for 15 min, followed by 85°C for 5 min to terminate reaction. Then RNase H (Solarbio, Beijing, China) was added into the mixture, which was incubated at 37°C for 30 min to degrade RNA.

The RNA-seq data were verified by qPCR using the same RNA samples used for RNA-seq. The concentration of total RNA was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). One μg of RNA was used as a template from which first-strand cDNA was synthesized using a SuperRT cDNA Synthesis Kit (CWBIO). QPCR was performed using an UltraSYBR Mixture Kit (CWBIO). The total volume of the qPCR mixture was 20 μL containing 10 μL of 2 × UltraSYBR Mixture (High ROX), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1 μL of cDNA, and 8.2 μL of ddH₂O. Reactions were carried out on a Bio-Rad iQ™5 Thermal Cycler (Bio-Rad) with the following reaction conditions: denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s, annealing and elongation for 1 min at 60 °C, and a melting curve process from 55 to 95 °C. Nine genes were selected as qPCR-verified targets. The primer sequences for these genes were listed in Additional file 8. Three biological replicates per groups and two technological replicates per sample were included in the qPCR. GAPDH was used as the endogenous reference gene, and the WSD2 group was set as the criterion. Expression levels of genes in each group were presented as expression folds relative to the criterion using the 2^-ΔΔCT method [67 (link)].

Total RNA of H9C2 cells was isolated using Ultrapure RNA Kit (Cat#: CW0581S, CWBio). 400 ng of RNA was subjected to reverse transcription-PCR with SuperRT cDNA Synthesis Kit (Cat#: CW0741S, CWBio) according to the instruction. Quantitative RT-PCR was performed with PCR primers listed in Table 1. UltraSYBR Mixture Kit (Cat#: CW2602M, CWBio) was employed to detect mRNA levels of these genes. All reactions were repeated 3 times and GAPDH was used to normalize target.
Table 1

List of utilized primers for qRT-PCR

Gene	Forward primer	Reverse primer
β1AR	CGACTGCTGGTGCTCGCGTCG	AGCGAAAGGGCAGCGTGATGGC
PGC-1α	CGCACAACTCAGCAAGTCCTC	CCTTGCTGGCCTCCAAAGTCTC
PGC-1β	CAAGAAGCGGCGGGAAA	GCTCATGTCACCGGAGAGATTT
VEGF	GCAGCGACAAGGCAGACTATT	ACCGTTGGCACGATTTAAGAG
NRF1	CCACGTTGGATGAGTACACG	CTGAGCCTGGGTCATTTTGT
ERRα	AAGCCCTGATGGACACCTC	GAAGCCTGGGATGCTCTTG
GAPDH	TGGAGTCTACTGGCGTCTT	TGTCATATTTCTCGTGGTTCA