Venom phosphodiesterase 1

LC-MS/MS for determination of m⁶A/A ratio was performed as previously described (56 , 57 (link)). Briefly, 1 μg of total RNA of whitefly adults was incubated at 95°C for 5 min and then on ice for 2 min. Two microliters of buffer [300 mM CH₃COONa, 2.8 M NaCl, and 10 Mm ZnSO₄ (pH 4.6)], 1 μl of nuclease S1 (180 U/μl; Sigma-Aldrich), and 1 μl of double-distilled water (ddH₂O) were added, and samples were incubated at 37°C for 4 hours. Subsequently, 10 μl of buffer 2 [0.5 M tris-HCl and 10 mM MgCl₂ (pH 9.0)], 5 μl of venom phosphodiesterase I (2 mU/μl; Sigma-Aldrich), and 1 μl of alkaline phosphatase (30 U/μl; Sigma-Aldrich) were added, and samples were incubated at 37°C for 2 hours. ddH₂O was then added to 200 and 10 μl of the solution injected into LC-MS/MS. Nucleosides were separated by reverse-phase ultraperformance liquid chromatography on a T3 C18 column (2.1 × 100 mm, 1.7 μm, Waters), with online mass spectrometry detection using a Sciex TripleTOF 6600 UPLC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside to base ion mass transitions of 282 to 150 (m⁶A) and 268 to 136 (A). Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running as part of the same batch of samples. The ratio of m⁶A to A was calculated on the basis of the calculated concentrations.

Global genomic DNA methylation was investigated using the electrospray ionization LC/MS method as previously described.⁵⁹ (link) Briefly, 500ng of genomic DNA was digested with nuclease P1 (Sigma-Aldrich #N8630), venom phosphodiesterase I (Sigma-Aldrich, #P-3243) and alkaline phosphatase (Sigma-Aldrich #P-4252) to yield individual nucleotides. Isotope labeled [¹⁵ (link)N3]2’-deoxycytidine and (methyl-d3, ring-6-d1)-5-methyl-2’-deoxycytidine internal standards were added to the digested DNA, then samples were run in the electrospray LC/MS. To determine percent methylation, we first calculated the mass of sample methylated cytosine by multiplying the ratio of the known mass of the labeled methylcytosine internal standard (ISmC) to ISmC peak area by the peak area of the unlabeled methylcytosine. We calculated the mass of the sample unmethylated cytosine by multiplying the ratio of the known mass of the labeled cytosine internal standard (ISC) to ISC peak area by the peak area of the unlabeled cytosine. Percent methylated cytosine was calculated as the mass of methylated cytosine divided by the sum of the masses of methylated cytosine plus unmethylated cytosine.

The nucleosides and modified nucleosides, including adenosine (A), guanosine (G), cytidine (C), uridine (U), N3-methylcytidine (m³C), 5-methylcytidine (m⁵C), N7-methylguanosine (m⁷G), N⁶-methyladenosine (m⁶A), N⁶,N⁶-dimethyladenosine (m^6,6A), 1-methyladenosine (m¹A), 1,2′-O-dimethyladenosine (m¹Am), and N⁶,2′-O-dimethyladenosine (m⁶Am) were purchased from various commercial sources and the detailed information of these nucleosides can be found in Supplementary Table S1 in Supporting Information. 1,N⁶-dimethyladenosine (m^1,6A) was synthesized in the current study. Venom phosphodiesterase I and TRIzol reagent were purchased from Sigma-Aldrich (Beijing, China). Calf intestinal alkaline phosphatase (CIAP) and S1 nuclease were obtained from Takara Biotechnology (Dalian, China). Proteinase K was obtained from Sangon Biotechnology (Shanghai, China). Dimethyl sulfate (DMS) was purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Trimethylsulfoniumiodide (Me₃SI) was purchased from Bide Pharmatech Ltd. (Shanghai, China). l-Methionine-(methyl-D₃) (D₃-Met) was purchased from Aladdin Industrial Inc. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium and fetal bovine serum were purchased from Thermo-Fisher Scientific (Beijing, China). Chromatographic grade methanol was purchased from Merck (Darmstadt, Germany).

Extraction of DNA was conducted through the standard phenol/chloroform/isoamyl alcohol [25:24:1 (v/v/v)] method with precipitation with 100% ethanol and 3 mol/L sodium acetate, pH 5.2. Following extraction DNA was re-dissolved in TE buffer. DNA quantification and 260/280 ratios were measured on a Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA). All DNA samples used had a 260/280 ratio equal to or greater than 1.80. DNA hydrolysis was performed as previously described.21 (link) In brief, 1 μg of genomic DNA was denatured by heating the sample at 100^°C for 3 minutes and subsequently chilled on ice. Next, 0.1 mol/L ammonium acetate (pH 5.3) and 2 units of nuclease P1 (Sigma, St. Louis, MO, USA) were then added to each sample. The mixture was incubated at 45^°C for 2 hours. Subsequently, 1 μL of 1 mol/L ammonium bicarbonate (pH 7.8) (Sigma) and 0.002 units of venom phosphodiesterase I (Sigma) were added to each sample. All samples were incubated for an additional 2 hours at 37^°C, then 0.5 units of alkaline phosphatase (Sigma) was added to the mixture and another 1 hour of incubation at 37^°C took place. The stable isotopes [¹⁵N₃]-2′-deoxycytidine and (methyl-d₃, ring-6-d₁)-5′-methyl-2′-deoxycytidine were then added to the samples to reach a final concentration of 1.00 and 0.25 ng/μL, respectively. The total volume for each sample was 35 μL at the end of hydrolysis.