S1 nuclease

Total m6A was measured in 1 μg of total RNA extracted from the uterus using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Shimadzu Corporation, Kyoto, Japan). RNA was heated at 95 °C for 5 min, followed by 2 min of cooling on ice. Subsequently incubated with 1 μL of S1 nuclease (Takara, Okasa, Japan) for 4 h at 37 °C. Then, 1 μL of alkaline phosphatase (Takara, Okasa, Japan) was added, and the reaction was incubated for 1 h at 37 °C. The reaction mixture was extracted with chloroform, freeze-dried at −40 °C for 24 h, and then dissolved in 100 μL of ultra-pure water post-centrifugation. HPLC separation was performed using a C18 column (Shimadzu Corporation, Kyoto, Japan) with a flow rate of 0.2 mL/min at 35 °C. Solvent A was 0.1% (vol/vol) formic acid in water, and solvent B was 0.1% (v/v) formic acid in methanol. A gradient of 5 min of 5% B, 10 min of 5–30% B, 5 min of 30–50% B, 3 min of 50–5% B, and 17 min of 5% B was used.

To reveal physical location of the bla_KPC-2 (and/or rmpA2) determinant in clinical isolates of K. pneumoniae, S1-Pulsed Field Gel Electrophoresis (S1-PFGE) was conducted, along with Southern blot as recently described [44 (link),45 (link)]. Overnight culture of different K. pneumoniae strains (Table S1) was imbedded in 1.0% agarose gel plugs (at the ratio 1:1), which was followed by the protease K treatment. Subsequently, S1 nuclease (Takara, Dalian, China) was applied to treat agarose gel plugs, prior to the separation of DNA fragments by PFGE [44 (link),45 (link)]. Then, Southern blot was routinely performed in accordance with the manufacturer’s protocol. In particular, two types of probes used here referred to digoxigenin-labeled PCR products separately targeting bla_KPC-2 and rmpA2.

S1-PFGE, for separating plasmids from chromosomes, was performed according to the method of Barton et al. (34 (link)), with modifications. Briefly, DNA plugs digested with S1 nuclease (Takara Bio, Shiga, Japan) were electrophoresed on a CHEF Mapper XA PFGE system (Bio-Rad, Hercules, CA) with autoalgorithms for 5 to 250 kbp. A lambda ladder (Promega, Fitchburg, WI) was used as the size marker. Extraction of the chromosome or plasmid DNA fragments from the electrophoresed gel for MiSeq analysis was performed using a Zymoclean large-fragment DNA recovery kit (Funakoshi Co., Ltd., Tokyo, Japan).

DsRNAs were extracted from 10 g of frozen mycelia by selective absorption to the columns of cellulose powder CF-11 (Whatman, UK) according to the method described by Morris and Dodds [25 (link)], with minor modifications. For the preparation of mycelium from strain GD-2, a mycelial agar plug 5 mm in diameter cut from a colony margin of a two-day-old culture was inoculated onto each PDA plate covered with a layer of cellophane membrane and cultured at 28 °C for 5 d. The harvested mycelia were stored at −80 °C until required.
After extraction, dsRNAs were further treated with DNase I and S1 nuclease (TaKaRa Bio Inc, China), which can digest the contaminated genomic DNA and ssRNA, respectively, and the quality of purified dsRNAs was then evaluated via 1.0% (w/v) agarose gel electrophoresis.

PCR-based replicon typing (PBRT) was performed on transconjugants and transformants using primers as described previously (Carattoli et al., 2005 (link)). Then PFGE with S1 nuclease (Takara Biotechnology, Dalian, China) digestion of whole genomic DNA was carried out as described previously (Barton et al., 1995 (link)). The resulting gels were analyzed by Southern transfer and probing with a DIG-labeled bla_CTX-M gene fragment according to the manufacturer’s instructions (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany).