Pentostatin

Total tRNA (∼100 ng) or purified tRNA isoacceptors were digested to single nucleosides by using 0.2 U alkaline phosphatase (Sigma-Aldrich), 0.02 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 0.2 U benzonase (Sigma-Aldrich) in Tris (pH 8, 5 mM, (Sigma-Aldrich)) and MgCl₂ (1 mM, (Sigma-Aldrich)) containing buffer. Furthermore, 0.5 μg tetrahydrouridine (Merck, Darmstadt, Germany), 1 μM butylated hydroxytoluene (Sigma-Aldrich), and 0.1 μg pentostatin (Sigma-Aldrich) were added to avoid deamination and oxidation of the nucleosides. The mixture was incubated for 2 h at 37°C and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g and 4°C for 30 min. 1/10 volume of SILIS (stable isotope labeled internal standard) as prepared in Borland et al. (53 (link)) was added to each filtrate before analysis by QQQ mass spectrometry.

Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-Deoxyadenosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG and 8-oxo-dA (see Supplementary Materials) were prepared according to the previously reported procedures [21 (link)]. Ultrapure water (18.3 MΩ.cm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA). Chloroform, methanol and n-hexane were purchased from Merck (HPLC-grade). Anhydrous sodium sulfate (Na₂SO₄) was purchased from Carlo Erba (Val de Reuil Cedex, France). All fatty acid methyl esters (FAME) used as reference standard for GC analyses were purchased from Sigma-Aldrich or Fluka (Steinheim, Germany) without further purification. Analytical silica gel thin-layer chromatography was performed on Merck silica gel 60 plates.

Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Chemicals for the synthesis of oligonucleotides were purchased from Sigma Aldrich, Fluka and Link Technologies. CuCl₂, L-methionine, L-ascorbic acid and alkaline phosphatase were purchased from Sigma-Aldrich. Hydrogen peroxide (30%) and solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-deoxyadenosine monohydrate and 2′-deoxyaguanosine were purchased from Berry & Associates Inc. (Dexter, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG, 8-oxo-dG and 8-oxo-dA (see Supporting Information) were prepared according to the previously reported procedures [31 (link)]. Ultrapure water (18.3 MΩcm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA).