Rna extract kit

For library construction, P. parasitica strain PpBS042 was firstly cultured on 5% CA solid medium with 0.01% CaCO₃ and 0.002% β-sitosterol, in darkness for 3–4 days (23°C). Then, the culture medium with the fresh mycelia were transformed into 5% CA broth with Petri solution for another 3 days. The total RNA was extracted from fresh mycelia of both the wild-type and PpAGO3^ΔRGG mutants by using the RNA extract kit (Aidlab, RN40). The RNA concentration and quality were examined by using NanoDrop 2000 and gel electrophoresis. Construction and sequencing of small RNA and RNA libraries were performed by Biomarker Technologies (Beijing, China) with Illumina novaseq 6000 platform. For construction of sRNA library, only 18–45 nt size small RNAs were used for sequencing. The wild-type and the two PpAGO3^ΔRGG mutants contain three biological replicates, respectively.

Total RNA from flower tissues was extracted using an RNA Extract Kit (Aidlab, Beijing, China) according to the manufacturer's instructions. DNase I (TaKara, Japan) was added to remove genomic DNA, and the samples were then subjected to cDNA synthesis using the Access RT-PCR System (Promega, USA) according to the manufacturer's instructions. The expression levels of McCHS, McDFR, McF3′H, McFLS, McANS, and McUFGT were analyzed using quantitative real-time PCR (RT-qPCR) with SYBR Green qPCR Mix (Takara, Japan) and Bio-Rad CFX96 Real-Time PCR Systems (BIO-RAD, USA), according to the manufacturers' instructions. The primers in this paper were designed by NCBI Primer BLAST and listed in Table 3.
qPCR analysis was carried out in a total volume of 20 µl containing 9 µl of 2×SYBR Green qPCR Mix (Takara, Japan), 0.1 µM specific primers (each), and 100 ng of template cDNA. The reaction mixtures were heated to 95°C for 30 s, followed by 39 cycles at 95°C for 10 s, 59°C for 15 s, and 72°C for 30 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products.

Total RNA was extracted from fresh leaf samples (50 mg) of eelgrass with RNA Extract Kit (Aidlab Biotechnologies Ltd., Beijing, China) according to the manufacturer’s instructions. The quality and integrity of total RNA were assessed by a NanoDrop (Thermo Fisher Scientific, Delaware, USA) and 1% agarose gel electrophoresis. The first-strand synthesis was carried out using the RQ1 RNase-free DNase I (Promega Corp., Madison, WI, USA) treated raw RNA (4 µg) as template and adaptor primer-oligo (dT) as primer (Table S1). The reaction were performed at 55 °C for 1 h and terminated by heating at 70 °C for 5 min. Products from this reaction were subsequently stored at −80 °C.