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ribonucleoside identities were confirmed by comparison of analyte retention times and MRM transitions to those of synthetic standards (i.e. those in the “digestion_mix_with_standards” sample”).MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phaseRibonucleoside analysis (25 – 200 ng on column) was performed using a Thermo Hypersil Gold aQ C18 column (100 × 2.1 mm, 1.9µm) on an Agilent 1290 Infinity II HPLC system with in-line UV detector (260 nm) for detection and quantification of unmodified ribonucleosides (A, U, G, C). Liquid chromatography was performed with a column temperature of 35 °C and a flow rate of 0.35 mL/min, with the following gradient: 0-4 min, 100% solvent A (0.1% (v/v) formic acid in water); 4–5.1 min, 0–1% solvent B (0.1% formic acid (v/v) in acetonitrile); 5.1-6.3 min, 1-6% solvent B; 6.3-7 min, 6% solvent B; 7-9 min, 6-50% solvent B; 9-11 min, 50-80% solvent B; and 11-15 min, 80% solvent B.Growth-dependent tRNA Reprogramming and Codon Bias Link Translation to Metabolic State in Enterococcus faecalis. 10.64898/2026.05.07.723122.Massachusetts Institute of TechnologyMichelle MitchenerBacteriaNot Applicablemass spectrometry assayCell pellets were resuspended in 1 mL of ice-cold TRIzol Reagent (Thermo Fisher, 15596026) and added to Lysing Matrix B tubes (MP Biomedicals, 116911100) on ice. Bacteria were lysed by bead-beating (3 x [40 s beat, 2 min rest on ice]) using a FastPrep-24 5G instrument (MP Biomedicals, 116005500) at a rate of 6.0 m/s. Samples were then centrifuged at 12000g at 4 °C for 5 min. The supernatants were transferred to new tubes to which 200 mL of chloroform was added. Samples were shaken vigorously for 15 s and then allowed to settle at ambient temperature for 2 min. The resulting mixtures were centrifuged at 12000g at 4 °C for 15 min, enabling separation of the aqueous (containing RNA) and organic (containing protein) phases. Large and small RNAs were isolated from a portion (200 µL) of the extracted aqueous phase by solid-phase extraction using a PureLink miRNA Isolation Kit (Thermo Fisher, K157001) according to the manufacturer’s protocol with minor modification. Prior to transfer to the first cartridge, ethanol was added to the aqueous phase to a final concentration of 15% (v/v). This ethanol concentration was determined empirically to minimize retention of small RNAs on the first cartridge while still ensuring complete retention of large RNAs on the first cartridge. Ethanol was added to the flowthrough of the first cartridge to give a final concentration of 70% (v/v), prior to addition to the second cartridge. Samples on both cartridges were washed twice with wash buffer, spun dry, and then eluted in 50 µL pre-warmed (70 °C), nuclease-free (non-DEPC-treated) water. RNA concentrations were determined using a NanoDrop spectrophotometer, and RNA quality and purity was assessed by Agilent 2100 Bioanalyzer pico and small RNA microfluidic chips. Large and small RNAs (500 ng – 1.25 µg) were digested with benzonase (0.25 U/µL, Sigma, E8263), alkaline phosphatase from bovine intestinal mucosa (0.1 U/µL, Sigma, P5521), and phosphodiesterase I (0.003 U/µL, Sigma, 3243) in a solution of 5 mM Tris (pH 8.0) and 2.5 mM MgCl2 supplemented with 0.1 mM deferoxamine (antioxidant, Sigma, D9533), 0.1 mM butylated hydroxytoluene (antioxidant, Sigma, W218405), 0.1 µg/mL coformycin (adenosine deaminase inhibitor, NCI, 27781713), and 50 nM [15N5]-2’-deoxyadenosine (for monitoring triple quadrupole instrument performance; not for quantitation purposes, Cambridge Isotope Laboratories, NLM-3895) in a total volume of 25 µL. Samples were incubated at 37 °C for 6 h. The digestion mixtures were then centrifuged at 13000g at 4 °C for 10 min and the supernatants subjected to LC-MS/MS analysis. Control samples included a “blank” sample (water only), a “digestion_mix_only” sample (containing the enzyme cocktail alone/no bacterial RNA), and a “digestion_mix_with_standards” sample (containing the enzyme cocktail plus a combination of synthetic modified ribonucleoside standards).Enterococcus faecalis OG1RFNot Applicablehttps://www.ebi.ac.uk/metabolights/MTBLS14602Michelle Mitchener. Singapore-MIT Alliance for Research and Technology. 1 Create Way, Enterprise Wing #03-14, Singapore 138602, Singapore. michelle.m.mitchener@icloud.com.Peter Dedon. Massachusetts Institute of Technology. Department of Biological Engineering, 25 Ames Street, 56-786, Cambridge, MA 02142, USA. pcdedon@mit.edu.Data were analyzed using Agilent MassHunter Workstation Software Quantitative Analysis (QQQ) (Quant-My-Way). Analyte peak areas were normalized to the sum of the UV signals (260 nm) for the unmodified nucleosides (A, U, G, C).RNA sizeTimepointBiological replicatemichelle.m.mitchener@icloud.comE. faecalis OG1RF overnight cultures were diluted into fresh BHI media and grown statically at 37 °C with ambient air for 6 h (n = 3 biological replicates), with samples taken every hour. Bacterial cultures (10-35 mL) were harvested by centrifugation at 5000g at 4 °C for 5 min. Cell pellets were then washed once with 35 mL of ice-cold PBS and pelleted again by centrifugation (5000g at 4 °C for 5 min). The wash supernatant was decanted, and the cell pellets were immediately lysed (see Extraction).MetabolomicsRNA modificationMetabolomicstargeted analysismultiple reaction monitoringAgilent softwareNot ApplicableAgilent 1290 Infinity HPLCSingapore-MIT Alliance for Research and TechnologyBacteriaEnterococcus faecalis OG1RFexperimental blank6495 Triple Quadrupole LC/MSMassHunter Quantitative AnalysisAgilent 6495 Triple QuadrupoleRNA modificationMetabolomicstargeted analysismultiple reaction monitoringAgilent softwareNot ApplicableAgilent 1290 Infinity HPLCSingapore-MIT Alliance for Research and TechnologyBacteriaexperimental blankEnterococcus faecalis OG1RF6495 Triple Quadrupole LC/MSMassHunter Quantitative AnalysisAgilent 6495 Triple QuadrupoleModified ribonucleoside identification and quantification was performed using an Agilent 6495 triple quadrupole mass spectrometer. The mass spectrometer was equipped with an Agilent Jet Stream electrospray ionization source and operated in positive mode with the following parameters: gas temperature, 120°C; gas flow, 11 L/min; nebulizer, 40 psi; sheath gas temperature, 400 °C; sheath gas flow, 12 L/min; capillary voltage, 1500 V; and nozzle voltage, 0 V. Dynamic multiple reaction monitoring (dMRM) was employed for analyte detection, with collision energies optimized for maximal sensitivity.falseRNA modification dynamics during Enterococcus faecalis OG1RF growthQuantitative analysis of relative levels of RNA modifications in small (primarily tRNA) and large (primarily 16S/23S rRNA) RNAs isolated from Enterococcus faecalis OG1RF across a growth time course in BHI media.2026-08-202026-05-28MTBLS14602