#METABOLOMICS WORKBENCH atsai_20250201_233304 DATATRACK_ID:5589 STUDY_ID:ST003726 ANALYSIS_ID:AN006112 PROJECT_ID:PR002314 VERSION 1 CREATED_ON February 13, 2025, 5:06 pm #PROJECT PR:PROJECT_TITLE Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals PR:PROJECT_TITLE a m7G in the Conserved P-loop of 23S rRNA PR:PROJECT_SUMMARY Extremophilic archaea employ diverse chemical RNA modifications, providing a PR:PROJECT_SUMMARY rich source of new enzymes for biotechnologically valuable RNA manipulations. PR:PROJECT_SUMMARY Our understanding of the modified nucleoside profiles in Archaea, as well as the PR:PROJECT_SUMMARY functions and dynamic regulation of specific RNA modifications is far from PR:PROJECT_SUMMARY complete. Here, we established an extensive profile of nucleoside modifications PR:PROJECT_SUMMARY in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS PR:PROJECT_SUMMARY analysis and rigorous non-coding RNA depletion, identifying - with high PR:PROJECT_SUMMARY confidence - at least four previously unannotated modifications in archaeal PR:PROJECT_SUMMARY mRNAs. Nucleoside quantification analysis conducted on total, large, small, and PR:PROJECT_SUMMARY mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus PR:PROJECT_SUMMARY kodakarensis revealed a series of modifications whose abundance is dynamically PR:PROJECT_SUMMARY responsive to growth temperatures, implying that specific RNA modifications are PR:PROJECT_SUMMARY fitness relevant under specific growth conditions. To predict the RNA-modifying PR:PROJECT_SUMMARY enzymes most likely to generate the new and dynamic RNA modifications, we PR:PROJECT_SUMMARY leveraged a bioinformatics analysis of open-access databases to annotate likely PR:PROJECT_SUMMARY functional domains of archaeal proteins. Putative enzyme activities were PR:PROJECT_SUMMARY confirmed in vitro and in vivo by assessing the presence of the target RNA PR:PROJECT_SUMMARY modification in genetic deletion strains of T. kodakarensis. Our approach led to PR:PROJECT_SUMMARY the discovery of a methyltransferase-encoded gene responsible for m7G PR:PROJECT_SUMMARY modification in the P-loop of 23S rRNA peptidyl transferase center and validates PR:PROJECT_SUMMARY a novel and effective platform for discovering RNA-modifying enzymes through PR:PROJECT_SUMMARY LC-MS/MS analysis that will accelerate efforts of the community towards PR:PROJECT_SUMMARY uncovering the complex and dynamic roles of RNA modifications. PR:INSTITUTE New England Biolabs PR:LAST_NAME Tsai PR:FIRST_NAME Yueh-Lin PR:ADDRESS 44 Dunham Ridge, Beverly, MA 01915 PR:EMAIL atsai@neb.com PR:PHONE 978-380-6587 #STUDY ST:STUDY_TITLE RNA modification profiles in archaeal and non-archaeal species ST:STUDY_SUMMARY Total RNA from five archaea, E. coli, S. cerevisiae and universal human ST:STUDY_SUMMARY reference RNA were digested to nucleosides and subjected to UHPLC-QqQ analysis. ST:STUDY_SUMMARY Each species was searched against a panel of 76 nucleoside standards. ST:INSTITUTE New England Biolabs ST:LAST_NAME Tsai ST:FIRST_NAME Yueh-Lin ST:ADDRESS 44 Dunham Ridge, Beverly, MA 01915 ST:EMAIL atsai@neb.com ST:PHONE 978-380-6587 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Thermococcus kodakarensis, Thermococcus sp. AM4, Methanococcus maripaludis, SU:SUBJECT_SPECIES Sulfolobus acidocaldarius, Sulfolobus islandicus, Saccharomyces cerevisiae, SU:SUBJECT_SPECIES Escherichia coli, Homo sapiens SU:TAXONOMY_ID 311400, 246969, 39152, 2285, 43080, 4932, 562, 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - E_coli_AT_01-r001 Species:Escherichia coli | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=E_coli_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - E_coli_AT_01-r002 Species:Escherichia coli | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=E_coli_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - Human_AT_02-r001 Species:Homo sapiens | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Human_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - Human_AT_02-r002 Species:Homo sapiens | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Human_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - Yeast_AT_03-r001 Species:Saccharomyces cerevisiae | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Yeast_AT_03-r001.d SUBJECT_SAMPLE_FACTORS - Yeast_AT_03-r002 Species:Saccharomyces cerevisiae | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Yeast_AT_03-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_rRNA_depl_rep1_AT_01-r001 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_rRNA_depl_rep1_AT_01-r002 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep1_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_total_rep1_AT_02-r001 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_total_rep1_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_total_rep1_AT_02-r002 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_total_rep1_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_rRNA_depl_rep1_AT_03-r001 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rep1_AT_03-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_rRNA_depl_rep1_AT_03-r002 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rep1_AT_03-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep1_AT_04-r001 Species:Sulfolobus acidocaldarius | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep1_AT_04-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep1_AT_04-r002 Species:Sulfolobus acidocaldarius | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep1_AT_04-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_rRNA_depl_rep2_AT_01-r001 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rep2_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_rRNA_depl_rep2_AT_01-r002 Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_rRNA_depl_rep2_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep2_AT_02-r001 Species:Sulfolobus acidocaldarius | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep2_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep2_AT_02-r002 Species:Sulfolobus acidocaldarius | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep2_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_rRNA_depl_rep2_AT_03-r001 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep2_AT_03-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_rRNA_depl_rep2_AT_03-r002 Species:Methanococcus maripaludis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_rRNA_depl_rep2_AT_03-r002.d SUBJECT_SAMPLE_FACTORS - M_mari_total_rep2_AT_04-r001 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_total_rep2_AT_04-r001.d SUBJECT_SAMPLE_FACTORS - M_mari_total_rep2_AT_04-r002 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=M_mari_total_rep2_AT_04-r002.d SUBJECT_SAMPLE_FACTORS - Mmari_Total_RNA-r001 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Mmari_Total_RNA-r001.d SUBJECT_SAMPLE_FACTORS - Mmari_Total_RNA-r002 Species:Methanococcus maripaludis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Mmari_Total_RNA-r002.d SUBJECT_SAMPLE_FACTORS - S_island_rRNA_depl_rep1_AT_01_r001 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rRNA_depl_rep1_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - S_island_rRNA_depl_rep1_AT_01_r002 Species:Sulfolobus islandicus | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_rRNA_depl_rep1_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - S_island_total_rep2_AT_02-r001 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_total_rep2_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - S_island_total_rep2_AT_02-r002 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_total_rep2_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_rRNA_depl_rep1-r001 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_mods_screen_01-r001.d SUBJECT_SAMPLE_FACTORS - TAM4_rRNA_depl_rep1-r002 Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_rRNA_depl_mods_screen_01-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_total_rep2_AT_02 Species:Thermococcus sp. AM4 | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_total_rep2_AT_02.d SUBJECT_SAMPLE_FACTORS - S_island_total_rep1_AT_01-r001 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_total_rep1_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - S_island_total_rep1_AT_01-r002 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_island_total_rep1_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep1_AT_02-r001 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep1_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - S_acid_total_rep1_AT_02-r002 Species:Sulfolobus islandicus | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=S_acid_total_rep1_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - TAM4_total_rep1_AT_03-r001 Species:Thermococcus sp. AM4 | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_total_rep1_AT_03-r001.d SUBJECT_SAMPLE_FACTORS - TAM4_total_rep1_AT_03-r002 Species:Thermococcus sp. AM4 | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=TAM4_total_rep1_AT_03-r002.d SUBJECT_SAMPLE_FACTORS - Tk_rRNA_depl_rep1_AT_01-r001 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_rRNA_depl_rep1_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - Tk_rRNA_depl_rep1_AT_01-r002 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_rRNA_depl_rep1_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - Tk_total_rep1_AT_02-r001 Species:Thermococcus kodakarensis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_total_rep1_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - Tk_total_rep1_AT_02-r002 Species:Thermococcus kodakarensis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_total_rep1_AT_02-r002.d SUBJECT_SAMPLE_FACTORS - Tk_rRNA_depl_rep2_AT_01-r001 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_rRNA_depl_rep2_AT_01-r001.d SUBJECT_SAMPLE_FACTORS - Tk_rRNA_depl_rep2_AT_01-r002 Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_rRNA_depl_rep2_AT_01-r002.d SUBJECT_SAMPLE_FACTORS - Tk_total_rep2_AT_02-r001 Species:Thermococcus kodakarensis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_total_rep2_AT_02-r001.d SUBJECT_SAMPLE_FACTORS - Tk_total_rep2_AT_02-r002 Species:Thermococcus kodakarensis | Sample source:Total RNA Genotype=Wild-type; RAW_FILE_NAME(Raw file name)=Tk_total_rep2_AT_02-r002.d #COLLECTION CO:COLLECTION_SUMMARY Universal human reference RNA (Agilent Technologies, Cat #740000) and CO:COLLECTION_SUMMARY Escherichia coli total RNAs (Thermo Fisher Scientific, Cat #AM7940) were CO:COLLECTION_SUMMARY purchased from commercially available sources. Saccharomyces cerevisiae (strain CO:COLLECTION_SUMMARY S288C) total RNA was obtained as described in Mulroney, L., Wulf, M.G., CO:COLLECTION_SUMMARY Schildkraut, I., Tzertzinis, G., Buswell, J., Jain, M., Olsen, H., Diekhans, M., CO:COLLECTION_SUMMARY Correa, I.R., Jr., Akeson, M., and Ettwiller, L. (2022). Identification of CO:COLLECTION_SUMMARY high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing. CO:COLLECTION_SUMMARY RNA 28, 162-176. 10.1261/rna.078703.121. Harvested archaeal cell pellets were CO:COLLECTION_SUMMARY resuspended in 10 mL of TRI reagent (Molecular Research Center, Inc., Cat CO:COLLECTION_SUMMARY #TR118). The resuspended cells were homogenized using a beads beater at 4.0 m/s CO:COLLECTION_SUMMARY for 20 seconds x 2 cycles (MP Biomedicals, FastPrep-24TM). Subsequently, the CO:COLLECTION_SUMMARY mixture was centrifuged at 14000 g for 5 minutes to precipitate any cell debris. CO:COLLECTION_SUMMARY Supernatants were collected post-centrifugation and treated with 50 μL of BAN CO:COLLECTION_SUMMARY reagent (Molecular Research Center, Inc., Cat #BN191) per mL of supernatant for CO:COLLECTION_SUMMARY aqueous-organic phase separation. RNA from the aqueous phase was isolated by CO:COLLECTION_SUMMARY isopropanol precipitation and subjected to DNase I treatment (NEB, Cat #M0303S) CO:COLLECTION_SUMMARY to remove genomic DNA contamination. To further purify the DNase I-treated RNA, CO:COLLECTION_SUMMARY an equal volume of acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo CO:COLLECTION_SUMMARY Fisher Scientific, Cat #AM9722) was added to the reaction and centrifuged at CO:COLLECTION_SUMMARY 21300 g for 2 minutes to separate the aqueous phase from the organic phase. The CO:COLLECTION_SUMMARY aqueous phase containing RNA was then precipitated with 1.5 volumes of CO:COLLECTION_SUMMARY isopropanol and 0.1 volume of sodium acetate (Sigma Aldrich, Cat #S7899) at CO:COLLECTION_SUMMARY –20°C overnight. Finally, the precipitated RNA pellets were washed with 75% CO:COLLECTION_SUMMARY ethanol and dissolved in nuclease-free water. CO:SAMPLE_TYPE Ribonucleic acid #TREATMENT TR:TREATMENT_SUMMARY To remove rRNA and tRNA, total RNA was separated into large (> 200 nt) and small TR:TREATMENT_SUMMARY RNA (< 200 nt) fractions using the RNA Clean and Concentrator Kit (Zymo TR:TREATMENT_SUMMARY Research, Cat #R1017). Subsequently, 50 μg of the large RNA fraction were TR:TREATMENT_SUMMARY subjected to rRNA depletion using the NEBNext rRNA Depletion Kit (NEB, Cat TR:TREATMENT_SUMMARY #E7850X) with the following changes: The NEBNext rRNA depletion solutions TR:TREATMENT_SUMMARY provided in the kit were substituted for customized DNA probe mixtures (at 1 uM TR:TREATMENT_SUMMARY for each probe) fully complementary to rRNA sequences corresponding to each TR:TREATMENT_SUMMARY archaeal species; All volumes for the probe hybridization, RNase H and DNase I TR:TREATMENT_SUMMARY digestion reactions were scaled up by fivefold in 10 parallel reactions. TR:TREATMENT_SUMMARY Following the enzymatic treatment, the reactions were cleaned up using RNA Clean TR:TREATMENT_SUMMARY and Concentrator Kit (Zymo Research, Cat #R1017), the mRNA-enriched fractions TR:TREATMENT_SUMMARY were eluted in 10 uL water and combined. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total RNA, RNA subfractions, and mRNA-enriched samples were digested to SP:SAMPLEPREP_SUMMARY nucleosides at 37°C overnight using a Nucleoside Digestion Mix (NEB, Cat SP:SAMPLEPREP_SUMMARY #M0649S). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A was at pH 4.5 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II UHPLC CH:COLUMN_NAME Waters XSelect HSS T3 XP (100 x 2.1mm, 2.5um) CH:SOLVENT_A 100% water; 10mM ammonium acetate CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT 1%-40% Solvent B in 26.5 min CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometric data were acquired using dynamic multiple reaction monitoring MS:MS_COMMENTS (DMRM) mode. Identification of each nucleoside species was based on the MS:MS_COMMENTS associated retention time and mass transition in the extracted chromatogram. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Integrative area under chromatographic peak MS_METABOLITE_DATA_START Samples E_coli_AT_01-r001 E_coli_AT_01-r002 Human_AT_02-r001 Human_AT_02-r002 Yeast_AT_03-r001 Yeast_AT_03-r002 M_mari_rRNA_depl_rep1_AT_01-r001 M_mari_rRNA_depl_rep1_AT_01-r002 M_mari_total_rep1_AT_02-r001 M_mari_total_rep1_AT_02-r002 S_acid_rRNA_depl_rep1_AT_03-r001 S_acid_rRNA_depl_rep1_AT_03-r002 S_acid_total_rep1_AT_04-r001 S_acid_total_rep1_AT_04-r002 S_acid_rRNA_depl_rep2_AT_01-r001 S_acid_rRNA_depl_rep2_AT_01-r002 S_acid_total_rep2_AT_02-r001 S_acid_total_rep2_AT_02-r002 M_mari_rRNA_depl_rep2_AT_03-r001 M_mari_rRNA_depl_rep2_AT_03-r002 M_mari_total_rep2_AT_04-r001 M_mari_total_rep2_AT_04-r002 Mmari_Total_RNA-r001 Mmari_Total_RNA-r002 S_island_rRNA_depl_rep1_AT_01_r001 S_island_rRNA_depl_rep1_AT_01_r002 S_island_total_rep2_AT_02-r001 S_island_total_rep2_AT_02-r002 TAM4_rRNA_depl_rep1-r001 TAM4_rRNA_depl_rep1-r002 TAM4_total_rep2_AT_02 S_island_total_rep1_AT_01-r001 S_island_total_rep1_AT_01-r002 S_acid_total_rep1_AT_02-r001 S_acid_total_rep1_AT_02-r002 TAM4_total_rep1_AT_03-r001 TAM4_total_rep1_AT_03-r002 Tk_rRNA_depl_rep1_AT_01-r001 Tk_rRNA_depl_rep1_AT_01-r002 Tk_total_rep1_AT_02-r001 Tk_total_rep1_AT_02-r002 Tk_rRNA_depl_rep2_AT_01-r001 Tk_rRNA_depl_rep2_AT_01-r002 Tk_total_rep2_AT_02-r001 Tk_total_rep2_AT_02-r002 Factors Species:Escherichia coli | Sample source:Total RNA Species:Escherichia coli | Sample source:Total RNA Species:Homo sapiens | Sample source:Total RNA Species:Homo sapiens | Sample source:Total RNA Species:Saccharomyces cerevisiae | Sample source:Total RNA Species:Saccharomyces cerevisiae | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:Total RNA Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:Total RNA Species:Sulfolobus acidocaldarius | Sample source:Total RNA Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:rRNA_depletion Species:Sulfolobus acidocaldarius | Sample source:Total RNA Species:Sulfolobus acidocaldarius | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:rRNA_depletion Species:Methanococcus maripaludis | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:Total RNA Species:Methanococcus maripaludis | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:rRNA_depletion Species:Sulfolobus islandicus | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:Total RNA Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Species:Thermococcus sp. AM4 | Sample source:rRNA_depletion Species:Thermococcus sp. AM4 | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:Total RNA Species:Sulfolobus islandicus | Sample source:Total RNA Species:Thermococcus sp. AM4 | Sample source:Total RNA Species:Thermococcus sp. AM4 | Sample source:Total RNA Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:Total RNA Species:Thermococcus kodakarensis | Sample source:Total RNA Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:rRNA_depletion Species:Thermococcus kodakarensis | Sample source:Total RNA Species:Thermococcus kodakarensis | Sample source:Total RNA N4-acetylcytidine_1 797 687 113083 110954 305732 303880 2728 2812 1456 1438 6081 6080 151829 147819 3247 2732 71710 71268 1685 1566 1298 1223 2266 1660 2697 2145 147735 147236 18988 17943 230268 345825 293573 339820 341812 925127 925191 27031 26893 789705 782993 11530 11740 1072522 1052258 N4-acetylcytidine_2 720 591 100586 99621 274378 271382 31 4 10 5595 5595 138293 136036 2434 1976 64175 64384 84 70 155 142 6 5 2491 2126 138926 137960 17996 17113 216906 318142 270948 312424 313502 845333 840582 24742 24622 710157 707619 11874 11868 942954 932292 N4-acetyl-2'-O-methylcytidine_1 7 7 1 8 15 4 2 3 2 3 58 57 1549 1560 50 50 914 940 2 1 1 0 3 3 19 1 2008 2016 77 53 2742 6043 5164 3331 3373 10799 10730 10 16 4878 4891 25 11 8752 8522 N4-acetyl-2'-O-methylcytidine_2 4 2 9 14 9 8 2 2 4 4 144 122 3634 3551 119 120 2629 2671 5 3 1 1 5 9 60 54 6679 6675 27 11 9134 29607 25008 17060 17122 54557 54400 78 75 14375 14436 55 46 21139 21284 N6-acetyladenosine_1 6 10 13 12 17 24 35 29 6 28 1181 1173 708 696 856 718 20 61 61 48 38 7 11 19 89 65 69 43 17 25 309 1580 1359 1677 1736 42 13 78 14 4 263 15 27 32 53 N6-acetyladenosine_2 5 5 16 15 28 56 14 16 6 1 14 13 18 14 5 19 10 16 14 54 21 20 10 9 21 11 39 48 24 10 25 99 59 114 50 73 52 20 28 34 57 4 7 45 66 3-(3-amino-3-carboxypropyl)uridine 874 874 210 159 56 4 801 370 412 397 197 140 370 391 23 22 21 257 22 23 287 21 3 9 471 507 594 538 14 12 315 13 61 380 493 57 493 39 869 33 696 273 261 662 829 2'-O-methyladenosine 20324 20291 11206653 10849280 7763638 7750816 322 370 431 280 8238 8207 381561 388003 5811 5086 128700 128777 171 193 117 173 1080 215 5401 4372 474859 473059 1526 1211 51717 801064 684601 1029565 1020654 283901 278527 4463 4349 350104 344501 2956 2867 479422 464037 5-carboxycytidine 733 652 3775 3843 3308 3792 11422 11010 18585 18678 19275 19698 17803 16214 8714 8164 11891 12026 22334 21239 16862 16939 20396 19099 20730 20674 17521 9131 9008 7180 7163 9067 8824 24882 26457 17466 17708 3441 3640 2'-O-methylcytidine 237859 231417 2472595 2445756 1529273 1512256 14792 14988 265534 265776 36703 37020 950526 933959 49 34 35 22 60 71 11 68 1420818 785059 12760 10468 1091766 1087131 37442 35848 241620 2291207 2018628 2423086 2429628 908443 900002 16152 16442 673132 666096 4169 3057 902911 895509 5-carboxymethyluridine_1 130 34 136 125 152 179 39 11 88 46 23 3 15 54 8 14 13 15 6 3 17 17 173 36 37 65 21 55 79 75 55 58 12 16 52 24 75 4 9 43 38 6 11 73 81 5-carboxymethyluridine_2 221 76 27 34 143 112 6 15 8 10 21 8 4 21 23979 21106 617151 614998 8150 7409 279793 264360 3 6 34 30 4 47 12 45 6 34 75 59 53 29 36 14 9 12 7 5-carboxymethylaminomethyluridine_1 28 12 4 21 21 6 24 10 25 146 35 15 7 4 27 2 15 14 14 2 148 25 568 300 9 7 5 29 22 29 126 11 12 74 16 523 418 39 46 239 257 4 10 288 288 5-carboxymethylaminomethyluridine_2 41 42 7 6 2 2 3 1 3 2 4 2 1 1 4 4 7 2 3 6 4 3 4 1 2 1 1 1 1 2 1 3 3 1 1 1 7 0 1 1 0 2 0 1 2 5-carboxymethylaminomethyluridine_3 25 44 10 5 4 21 2 3 50 3 43 4 9 2 17 25 14 6 2 3 49 41 90 41 15 3 8 10 8 13 8 5 6 6 5 80 96 7 2 76 67 4 7 7 13 dihydrouridine 30282 28705 1885 1974 196645 200814 89 115 80 93 66 48 15 36 115 124 124 60 66 34 90 67 57 111 129 144 64 30 204 205 72 39 54 21 83 36 48 359 269 88 94 1019 1129 97 127 5-formylcytidine 3269 3125 595 664 0 9 986 1037 833 866 36 51 479 431 165 163 344 374 536 567 746 825 1385 857 635 521 1093 1115 6 4 571 924 932 1147 1183 1032 1102 1095 1117 1577 1497 232 267 1501 1422 N6-formyladenosine_1 27 4 5 10 14 2 16 28 1136 1107 8 12 23 7 19 12 25 7 26 13 980 954 2129 1023 16 25 4 13 3 8 31 27 52 43 12 11 11 37 26 1 5 6 9 11 N6-formyladenosine_2 10 27 62 91 21 31 13 28 929 944 10 8 28 6 16 3 9 1 10 16 832 778 1790 879 19 6 13 5 326 34 155 9 39 3 47 4 10 6 1 5 97 3 1 195 2 2'-O-methylguanosine 219884 209247 1652098 1626846 1119494 1105374 3913 3880 79507 78608 13495 13746 391882 379984 6982 6150 129359 128422 1402 1237 55228 51116 172386 87400 6227 4992 325686 328704 6940 6544 131412 482120 415280 726665 723707 485410 485845 11016 11079 588192 588345 5757 5408 824952 805692 5-hydroxymethylcytidine 42 78 194 162 14 13 8 3 4 4 8 5 236 202 63 14 64 105 26 60 10 10 3 2 12 6 22 29 12 8 15 3 6 493 507 95 142 4 3 24 18 7 29 20 N6-hydroxymethyladenosine_1 145 218 115 109 178 187 57 56 30 33 20 13 35 18 10 11 37 54 14 54 35 30 97 62 7 47 20 39 6 17 11 140 97 292 315 135 136 51 75 86 61 65 50 115 115 N6-hydroxymethyladenosine_2 3216 3078 1928 1813 2840 2814 799 786 423 413 176 200 306 313 108 84 146 156 288 259 214 193 1633 853 250 197 176 184 37 5 141 2100 1784 4856 4830 1556 1519 937 881 886 897 762 639 1429 1394 5-hydroxyuridine 380 375 48 45 50 2 36 22 96 80 83 67 117 128 35 21 19 6 28 12 375 271 1088 580 15 5 9 30 4 11 17 28 32 77 9 6 1 116 136 53 62 6 6 13 7 N6-isopentenyladenosine_1 13953 13442 10916 10849 1511102 1496435 90 110 69 79 96 90 84 79 133 141 119 129 129 142 138 124 1341 1304 103 96 109 96 9853 11548 107 479 546 386 413 506 243 232 116 125 240 227 143 155 N6-isopentenyladenosine_2 15802 15215 12211 12022 1711669 1688050 100 94 82 81 109 105 102 84 129 162 170 149 142 160 181 139 1416 1461 123 77 119 124 10924 10883 115 531 482 463 499 596 260 254 123 152 267 249 159 155 2'-O-methylinosine_1 17 10 12 17 8 26 3 13 3 2 13 10 13 12 2 2 8 21 3 11 9 4 11 6 3 6 12 3 6 43 5 17 19 10 4 8 23 20 17 20 19 3 4 29 39 2'-O-methylinosine_2 21 51 3 22 17 11 16 5 66 13 2 3 5 13 6 6 6 4 8 14 6 17 17 3 6 7 41 33 9 36 19 36 36 49 50 34 15 24 5 70 38 5 13 27 28 4-demethylwyosine 5 4 29 32 603 572 861 861 32681 32625 36 35 538 513 556 556 17020 17055 5 2 6 3 54709 27184 30 26 3744 3743 5 4 3504 7465 6318 1516 1505 6139 6093 isowyosine 4 11 10 19 9 10 3 12 5 5 1292 1300 27267 26442 17 9 386 392 536 475 38089 35905 6 11 407 332 45019 44539 16 6 885 89237 75290 63204 63450 2470 2390 N6-(cis-hydroxyisopentenyl)adenosine 8 5 18 6 21 10 1 9 5 4 4 2 4 2 6 1 3 5 7 2 2 4 5 2 2 4 2 2 1 8 2 46 3 8 46 3 4 9 9 1 14 8 16 5 13 1-methyladenosine 7630 7443 2435829 2406791 19563922 19185572 37230 36150 2193255 2193367 226279 214932 6156575 6098579 274528 240089 5761521 5841615 42948 39589 2952728 2827567 8414074 4585223 102177 83896 9308106 9338662 37042 35080 3185220 18638480 17215892 20006377 20089275 9952947 9871337 56035 56966 6336309 6354040 3722 3670 6733463 6702759 1,2'-O-dimethyladenosine 3 5 144 161 2 10 19 24 9 27 25 23 148 116 5 8 105 95 5 3 5 3 1 9 6 15 408 423 6 3 65 809 745 472 583 244 235 2 5 24 39 2 7 48 34 1-methylguanosine 789842 758707 61964 61122 2787294 2778046 815 729 63274 63014 2879 2959 118305 114588 1046 861 63833 62819 281 325 51474 48190 357390 180028 402 282 106402 105333 1306 1176 61043 300903 256901 293142 290954 228599 225025 1078 1115 160353 157562 836 904 223206 216619 1-methylinosine 13 19 5535 5417 150095 146505 3677 3743 135719 134354 16354 16388 353210 344640 10228 8749 208528 207251 1698 1537 122709 114258 673905 347722 2385 1908 244310 241347 1881 1799 95669 653055 556096 880998 873483 326878 326296 1596 1512 278288 276689 1704 1817 402107 391547 1-methylpseudouridine_1 72 55 453 324 560 680 733 799 21867 22048 70 91 189 205 18 50 34 31 414 159 16979 16235 32848 16672 114 89 191 259 43 43 1618 292 289 380 441 5235 5182 18 25 6389 6259 414 811 8717 9861 1-methylpseudouridine_2 19 8 178 191 495 535 475 467 17431 17550 26 56 146 156 6 22 25 35 197 189 13542 13022 25757 13188 68 52 164 193 14 10 1230 223 201 279 313 4241 4358 19 35 5240 5411 20 31 7311 7522 N2,N2,7-trimethylguanosine_1 11 7 214 186 120 115 5 9 13 4 10 3 3 60 59 80 6 92 32 186 102 35 82 77 18 1 6 10 1354 459 12 42 37 114 13 5 16 39 2 5 4 199 186 9 9 N2,N2,7-trimethylguanosine_2 12 3 351 308 124 142 4 4 4 9 9 2 11 7 3241 2914 105735 105100 225 230 37961 35661 2 1 12 18 5 56 45 46 53 7 13 3 2 8 1 3 2 11 3 N2,N2-dimethylguanosine 400 572 21814 21195 1883185 1873074 2035 2051 153185 152873 13485 13509 412157 403431 15 1 221 198 1 1 2 1 605410 304683 2651 2169 420870 418268 1072 1010 74231 1076028 915869 914494 916882 226995 223998 2457 2345 261958 257507 3870 3697 372635 364984 N2,7-dimethylguanosine_1 14 59 30 6 196 119 3 7 5 3 7 7 16 11 319 306 915 909 1 8 3 4 4 34 10 19 13 4456 4156 1306 22 18 53 13 5682 5830 18 59 4516 4593 15 14 5468 5272 N2,7-dimethylguanosine_2 8 19 3 3 13 21 3 1 2 1 3 11 2 4 6 14 7 3 10 22 2126 2004 4 2 4 1 2043 19 16 2 4 9425 9228 90 76 7293 7603 12 7 8998 8933 2,8-dimethyladenosine 6 6 1 1 4 8 4 2 4 1 5 5 5 5 6 8 8 30 2 16 1017 961 8 15 8 2 5 5 19 20 52 4 6 2 8 2 13 10 4 4 5 4 1 6 7 2-methyladenosine 2286405 2205248 15 101 67 155 75 45 19 23 118 79 66 32 17 7 688 662 15 1 3 4 46 20 49 91 27 51 77 58 20 58 56 131 105 94 128 77 168 106 96 43 38 10 7 N2-methylguanosine 507147 486940 35536 34600 594975 589927 618 653 59868 59194 11906 11805 250729 251039 1 6 2 1 3 2 3 1 163311 82479 1004 893 158638 159514 491 366 39998 334422 286458 431616 427186 101676 99695 1082 1092 103679 104136 468 612 141746 138873 N2,2'-O-dimethylguanosine 2 3 5 6 4 1 2 1 1 5 56 54 707 659 8080 7276 195421 194944 1782 1615 23248 22269 2 0 2 4 26 35 115 92 8877 114 89 860 854 29917 29809 250 230 24120 23997 285 340 43625 42360 3-methylcytidine 1104 1058 50671 49169 1700105 1677752 4 6 172 145 1578 1362 3 4 6 4 5 9 6 8 15 51 1117 892 7319 7403 14 21 140 15821 14173 5701 6383 705 845 96 122 258 17 11 158 24 3-methylpseudouridine_1 21268 20712 19 65 52 30 41 38 2859 2859 57 19 40 86 212 196 6726 6686 3 4 11 5 6185 3155 12 54 3 34 10 61 902 14 5 16 3 2873 2845 28 35 4149 3978 450 68 5804 5440 3-methylpseudouridine_2 9891 9735 18 33 10 18 5 4 1377 1354 3 22 15 17 7849 7016 190793 189901 1642 1512 22803 21662 2896 1512 2 18 14 16 12 6 450 31 29 8 6 1445 1454 22 27 2038 2071 17 45 2817 2819 3-methyluridine 29768 28877 16776 16416 31948 31703 8 7 15 10 5 2724 2581 200 161 6665 6577 3 1 2 12 9 5 5 12 3026 2966 12 15 1446 2242 1891 4837 4824 4426 4492 20 24 6238 6105 20 3 9078 8871 3,2'-O-dimethyluridine 2 6 5 4 11 14 2 1 3 2 15 9 3 7 23 15 26 34 29 139 1536 1536 6 14 3 6 3 3 7 3 7 5 11 4 5 4 21 5 10 6 5 5 45 4 46 N4,N4-dimethylcytidine 38 8 26 13 53 11 14 8 15 11 2 3 2 18 15 53 47 74 49 26 10 16 11 31 12 2 6 17 1229 1060 18800 17 7 5 16 78654 78470 85 134 61344 61292 54 40 87612 85982 N4,N4,2'-O-trimethylcytidine 4 12 5 30 27 3 57 12 21 68 5 7 59 27 29 17 15 5 147 111 101 98 23 9 5 24 8 6 7 4 20 220 252 502 270 121 21 6 10 4 21 4 11 6 1 N4-methylcytidine_1 259190 251479 135985 135099 1052421 1044456 3877 4005 27551 27849 14855 14938 367096 358383 3 5 1 0 2 1 2 3 120320 61367 3454 2837 292005 288445 14978 14303 154665 704759 611496 872657 878232 578761 575605 12771 13118 470179 475223 3186 2933 618029 610386 N4-methylcytidine_2 35 33 145 134 2 6 15 2 5 2 1 6 12 3 2845 2384 38575 38037 1038 834 6659 6373 6 1 6 2 8 6 9 9 3 6 18 14 12 2 19 N4,2'-O-dimethylcytidine 155128 149799 278 283 8 5 6 1 1 8 211 186 8804 8599 45 30 50 42 18 7 38 21 1 2 74 70 10783 10718 14 4 486 26896 23081 28531 28399 1668 1495 22 17 324 361 20 5 485 444 5-methylcytidine 252042 244498 133232 132245 1025055 1022605 3839 3833 26938 27006 14533 14752 357051 348258 16 10 8 15 19 37 14 4 118475 59419 3423 2852 286998 282445 14457 14036 151659 693228 598425 847239 853638 562661 561063 12390 12857 461772 464159 3058 2790 602788 600199 5,2'-O-dimethylcytidine 154975 149313 247 260 8 17 2 3 1 2 253 230 8874 8447 100 78 104 117 109 89 89 106 4 13 62 65 10768 10543 23 30 456 27212 23031 28489 28818 1475 1590 15 16 336 380 2 1 500 498 5-methyldihydrouridine 121 126 103 64 415 295 19 16 2122 1925 3 2 87 21 3 1 11 1 0 1 1 3 21054 11546 60 21 31 65 7 2 1376 2 8 4 4 6589 6501 49 51 5627 5578 65 6 6812 6646 5-methyl-2-thiouridine 26 21 21 26 102 47 17 8 10 9 15 19 36 34 3038 2741 39858 39774 225 208 114 130 11 4 48 211 332 90 123 83 15833 181 129 188 202 30074 30024 52 57 30441 30743 147 412 39269 40055 5-methyluridine 24696 23744 860 841 67883 68038 249 242 143 131 4 1 22 21 2753 2389 38568 38117 973 836 6733 6290 183 131 47 45 202 197 31 22 1152 270 243 38 22 1689 1645 459 505 2396 2439 343 336 2771 2906 5,2'-O-dimethyluridine 38 18 1062 1100 2 2 5 1 9 2 4 5 4 7 3279 2840 199755 198926 833 757 112557 106055 2 3 7 3 10 5 2 5 1 27 23 7 9 2 5 12 5 12 4 4 19 3 16 N6,N6-dimethyladenosine 5398716 5223266 3226637 3174374 3984606 3965995 11696 11561 725831 722939 11712 11777 226748 219857 23 5 31 7 17 20 3 43 3955501 2001745 22720 18102 386939 382118 80561 71408 428722 563212 476116 862078 860589 1303577 1285095 4399 4268 906901 887797 5653 5263 1189805 1152082 N6,N6,2'-O-trimethyladenosine 1 3 1 2 13 1 2 4 2 3 2 1 2 8 2 6 2 1 1 7 5 2 2 2 5 3 1 2 1 1 1 2 6 2 1 9 1 2 3 1 1 3 9 0 1 N6-methyladenosine 3533318 3430745 2320100 2289233 111642 111227 1742 1740 7013 6984 4025 4044 88839 89987 3 4 3 1 1 14 26 21 40977 22747 7256 5771 123415 122867 2509 2214 7851 295534 248540 259596 258485 35898 35447 14727 14480 33989 33034 14660 13940 46687 44776 N6,2'-O-dimethyladenosine 197 122 75847 74637 96 119 11 24 4 18 6 31 24 41 12 5 11 7 5 20 7 21 300 198 10 12 71 56 2558 2751 44 176 155 133 122 130 111 50 17 148 139 43 134 221 264 O6-methylguanine 76 76 40 30 26 55 7 12 17 25 13 21 14 15 14 18 7 5 24 24 1 27 7 9 17 10 27 22 48 61 7 79 7 57 13 33 54 48 8 14 7 13 34 20 34 N6-methyl-N6-threonylcarbamoyladenosine_1 437 447 35 16 33 345 9 7 3 20 9 13 4 14682 12726 138373 138339 9966 8990 1177264 1107305 4 3 10 7 63 63 3 6 158 10 12 9 3 14 33 10 8 7 18 12 213 233 216 N6-methyl-N6-threonylcarbamoyladenosine_2 638 616 96 87 4 1 1 4 1 1 0 2 1 3 0 8 2 1 1 4 1 1 1 2 0 5 1 1 2 1 0 2 7 3 8 2 3 3 2 2 2 1 3 2 2 7-methylguanosine 6389987 6207940 1201123 1191337 7804199 7671824 295 286 90 105 3005 2995 48398 47873 10 8 16 14 9 10 7 7 483 313 11554 9757 104615 103326 5900 5483 48999 216776 194348 172696 172341 295030 290415 7046 7241 160858 155719 657 663 209975 207381 8-methyladenosine 3572210 3430775 2309287 2279568 111131 110593 1643 1678 6970 7022 4100 4151 89089 89944 10 1 4 14 9 7 3 5 39412 23727 7171 5839 123937 123128 2324 2108 8077 294716 249072 258663 258503 35585 35270 14957 14276 33966 33068 14460 13995 46548 44885 5-methoxycarbonylmethyl-2-thiouridine_1 12 15 411 346 50657 50239 2 4 7 7 44 56 1346 1340 23 32 609 590 23 35 23 5 12 13 44 264 200 86 48 17 442 414 2261 2192 126 20 30 31 12 10 11 13 16 12 5-methoxycarbonylmethyl-2-thiouridine_2 29 29 724 786 98916 99166 12 18 7 21 58 69 1890 1868 23 7 1393 1416 2 10 17 14 10 44 15 15 590 525 270 451 58 806 682 3930 3857 2 20 43 12 21 47 24 17 3 22 5-methoxycarbonylmethyluridine_1 41 7 9 10 19825 19995 2 37 20 13 6 44 46 50 50 16 34 48 14 39 59 53 8 2 12 62 339 324 7 11 14 786 582 106 95 24 528 17 22 86 30 72 37 10 36 5-methoxycarbonylmethyluridine_2 18 13 52 20 26604 26482 37 55 18 27 11 10 46 52 7 17 25 14 35 41 36 80 8 11 15 68 568 535 10 16 25 822 673 96 114 16 53 47 54 206 49 112 12 222 47 5-methoxycarbonylmethyl-2'-O-methyluridine 11 12 24 9 21 16 1 3 16 8 14 14 285 319 9 1 90 112 7 15 15 20 7 5 17 6 2134 2208 36 13 11 6050 5139 428 421 9 13 10 7 12 9 2 2 5 1 uridine 5-oxyacetic acid methyl ester_1 12313 12110 13 33 5 24 48 24 2 31 67 7 14 5 111 26 11 124 68 71 32 291 5 4 7 22 891 616 14 13 110 107 69 270 37 30 340 32 23 546 507 10 48 71 46 uridine 5-oxyacetic acid methyl ester_2 10879 10560 13 13 6 7 32 18 7 16 14 13 32 45 12 2 5 2 17 4 10 8 5 6 6 5 2 15 6 51 15 12 8 11 6 11 14 5 3 16 13 2 6 13 2 uridine 5-oxyacetic acid methyl ester_3 42339 40708 25 27 14 20 22 24 13 8 7 8 5 5 266 16 18 7 95 15 24 8 5 7 39 53 9 81 2 21 5 26 19 9 6 4 38 15 17 175 481 43 50 144 899 5-methylaminomethyl-2-thiouridine_1 9346 8944 2 11 4 2 65 66 5365 5381 8 7 56 17 12 5 8 27 33 5564 5359 5521 2789 2 6 2 22 6 118 95 5-methylaminomethyl-2-thiouridine_2 13456 13058 5 11 15 2 70 88 7838 7775 8 5 5 1 1 0 11 3 12 28 8041 7668 8086 3983 2 2 3 7 9 4 4 2 2 3 5 16 5 6 14 8 2 6 15 5 11 5-methoxyuridine 10 5 10 16 12 13 27 12 9 7 4 3 2 8 96 88 21 38 254 261 25 22 14 9 59 49 55 111 2 1 397 3 10 52 22 38 30 26 36 70 38 163 172 211 936 2-methylthio-N6-isopentenyladenosine_1 448285 430579 63750 61649 151 144 80 71 88 73 75 80 66 68 45 19 15 26 73 66 54 32 89 39 44 40 44 49 52 51 37 94 80 43 47 47 169 148 154 154 131 97 92 80 2-methylthio-N6-isopentenyladenosine_2 425202 406261 60834 58693 88 186 1908 1809 1667 1628 1622 1625 1730 1721 5719 5518 4923 4735 4274 4156 4052 4009 12 54 3938 3768 3338 3229 152 108 3415 4038 4047 3878 3799 3522 128 155 98 139 57 59 43 48 2-methylthio-N6-methyladenosine 3 6 7 3 12 4 1 3 3 0 2 6 2 2 11 4 3 4 0 5 4 10 5 3 15 10 4 2 3 4 0 4 6 8 1 2 2 5-carbamoylmethyluridine_1 7 17 232 193 19636 19621 8 2 834 815 13 8 9 15 18 12 4 6 8 2 1062 1024 3736 1896 17 8 16 5 2 4 8 19 12 30 31 28 8 12 2 24 11 754 656 3 1 5-carbamoylmethyluridine_2 4 16 200 203 17556 17586 1 1 87 82 2 1 3 2 1 3 2 1 3 3 108 110 339 150 2 3 3 2 1 2 14 2 1 2 1 9 23 5 6 1 14 19 16 6 9 pseudouridine 70647 70318 240133 242712 267761 276143 31 63 13133 12794 20 111 12892 12899 135 99 5847 5896 21 20 12150 12415 49939 27743 199 219 34969 35105 184 182 13802 58738 56010 30003 30998 53142 54002 42 62 38743 40658 77 115 47386 47865 adenosine 87338199 89612269 73582212 72118132 82714799 81062185 35095030 35274383 17344237 17434281 11593943 11640309 13467890 13339110 7955281 7243676 6108618 6056761 20423151 18645404 15338478 14673681 51147016 37807643 16678496 14137797 13422221 13379182 6984792 6685348 6310019 17391537 15514827 23563475 23455668 18105429 17992145 17738828 17556334 16927494 16742475 18195139 16930359 19701987 19220919 cytidine 10268942 10096934 10484868 10546153 8941861 9057212 2201219 1962298 2109890 2191992 1336839 1355271 2584488 2649873 832897 790068 1446575 1461290 1933155 1860312 2177260 2176112 6717120 5159652 2095056 1935020 3169466 3198731 1219842 1200761 1415571 3787630 3821964 4798838 4916742 4197346 4230291 3182420 3299014 3408570 3532451 436527 418788 3801514 3752329 guanosine 31350501 30613965 28204448 27490994 24938383 24623781 9509406 9666667 7791813 7801878 3183523 3200921 7037898 6882888 1769007 1619824 2912418 2915122 4768995 4425975 6459426 6175783 14699400 9119940 4377618 3820337 7488634 7468573 918601 875541 3842683 8483557 7474343 11358866 11364648 9421987 9359494 7523958 7692467 10518873 10561009 4086214 3636193 12736875 12631387 inosine 5184 5114 23994 23200 461854 456227 601 646 2817 2710 156 160 1167 984 353 1223 486 684 1223 963 3043 3045 1635 818 1003 1048 1671 1411 176 197 432 1354 1531 890 671 601 661 2644 2809 7171 7114 543 311 10587 10664 uridine 3929713 3833026 2849157 2762181 4098158 3927804 2412152 2338769 1135252 1150212 787995 792828 884618 928751 482869 450756 377135 376827 1317077 1230509 1032555 1018575 2087574 1254172 1302185 1181703 1025153 1027716 83822 80218 359732 927329 893110 1365143 1379791 934850 903493 1119155 1158587 1002500 1036093 278047 266868 1190732 1185090 2-thiocytidine 10976 10671 5 9 6123 6160 375 388 35449 35308 14 9 17 18 2 6 5 4 148 170 28715 27119 382595 213401 2 7 21 4 895 933 22771 1876 1573 5 11 121233 120347 708 759 97459 99715 108 77 123818 120542 2-thiouridine 442 419 24 44 9 12 96 72 1020 976 592 587 12107 11986 296 273 6410 6300 7 33 816 808 1976 970 22 6 258 280 7 4 5126 309 309 18415 18777 13869 13533 3 8 18702 18908 5 26 22783 22497 2-thio-2'-O-methyluridine 13 6 31 7 27 6 28 20 9 12 39 53 759 806 23 10 320 293 11 17 6 12 8 3 16 40 274 266 5 20 21 818 654 900 958 6 20 45 12 27 9 21 33 5 8 4-thiouridine 6450 6369 4 8 14 21 49 77 10374 10002 588 691 915 880 9 3 582 513 27 25 7798 7323 13712 6991 81 74 9580 9039 51 74 5139 12171 10320 1209 1078 14182 14146 113 125 19182 19592 62 42 23445 22369 N6-threonylcarbamoyladenosine_1 2731 2629 656 618 33762 33307 103 91 3130 3108 305 300 11992 11571 66 84 4763 4607 43 38 1925 1832 2921 1452 65 50 10095 10050 186 316 721 24111 20528 20885 20903 2043 1949 21 37 236 216 14 19 317 309 N6-threonylcarbamoyladenosine_2 4229 4067 974 973 50424 50639 150 179 4718 4725 499 459 17897 16998 57 108 6745 7030 79 17 2855 2644 4437 2251 110 66 15319 15147 523 458 969 34518 29255 30262 29600 2958 2838 62 51 315 286 16 25 379 456 2'-O-methyluridine 24954 24130 196791 193645 109904 108222 327 337 11991 11875 1569 1566 62408 60919 691 594 21629 21643 122 108 8817 8307 13835 7020 948 756 50714 50883 546 532 13601 78658 66400 103688 103206 47587 47296 1994 1969 56098 56777 956 1024 91496 90629 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention_Time (min) Precursor Ion Product Ion N4-acetylcytidine_1 6.56 286.11 154.1 N4-acetylcytidine_2 6.56 286.11 112.1 N4-acetyl-2'-O-methylcytidine_1 9.83 300.11 154.1 N4-acetyl-2'-O-methylcytidine_2 9.83 322.1 176.1 N6-acetyladenosine_1 7.77 310.11 178.2 N6-acetyladenosine_2 7.77 332.1 200.2 3-(3-amino-3-carboxypropyl)uridine 1.63 346.1 214.1 2'-O-methyladenosine 8.99 282.1 136.1 5-carboxycytidine 0.96 288.11 156.1 2'-O-methylcytidine 3.59 258.1 112.1 5-carboxymethyluridine_1 1.48 303.1 171.1 5-carboxymethyluridine_2 1.48 303.1 125.1 5-carboxymethylaminomethyluridine_1 1.1 332.1 200.1 5-carboxymethylaminomethyluridine_2 1.1 332.1 125.1 5-carboxymethylaminomethyluridine_3 1.1 354.1 222.1 dihydrouridine 0.913 247.11 115 5-formylcytidine 4.52 272.11 140.1 N6-formyladenosine_1 9.11 296.1 164.1 N6-formyladenosine_2 9.11 296.1 136.1 2'-O-methylguanosine 6.05 298.1 152.1 5-hydroxymethylcytidine 1.56 274.11 142.1 N6-hydroxymethyladenosine_1 8.34 298.11 166.1 N6-hydroxymethyladenosine_2 8.34 298.11 136.1 5-hydroxyuridine 1.37 261.1 129.1 N6-isopentenyladenosine_1 26.21 336.1 136.1 N6-isopentenyladenosine_2 26.21 336.1 204.2 2'-O-methylinosine_1 5.92 283.11 137.1 2'-O-methylinosine_2 5.92 305.1 159.1 4-demethylwyosine 11.85 344.1 212.1 isowyosine 16.03 336.1 204.2 N6-(cis-hydroxyisopentenyl)adenosine 15.65 352.1 220.2 1-methyladenosine 2.35 282.1 150.1 1,2'-O-dimethyladenosine 4.36 296.1 150.1 1-methylguanosine 6.11 298.1 166.2 1-methylinosine 5.89 283.1 151.1 1-methylpseudouridine_1 2.15 259.11 223.1 1-methylpseudouridine_2 2.15 259.11 139.1 N2,N2,7-trimethylguanosine_1 7.81 326.1 196.2 N2,N2,7-trimethylguanosine_2 7.81 326.1 194.2 N2,N2-dimethylguanosine 8.46 312.1 180.2 N2,7-dimethylguanosine_1 6.17 312.1 182.1 N2,7-dimethylguanosine_2 6.17 312.1 180.1 2,8-dimethyladenosine 13.63 296.1 164.1 2-methyladenosine 10.06 282.1 150.2 N2-methylguanosine 6.59 298.1 166.2 N2,2'-O-dimethylguanosine 10.79 312.11 166.1 3-methylcytidine 1.93 258.1 126.1 3-methylpseudouridine_1 2.89 259.11 223.1 3-methylpseudouridine_2 2.89 259.11 139.1 3-methyluridine 5.15 259.11 127.1 3,2'-O-dimethyluridine 9.59 273.11 127.1 N4,N4-dimethylcytidine 5.84 272.1 140.2 N4,N4,2'-O-trimethylcytidine 8.5 286.1 140.2 N4-methylcytidine_1 2.74 258.1 126.1 N4-methylcytidine_2 2.74 258.1 95 N4,2'-O-dimethylcytidine 5.44 272.1 126.1 5-methylcytidine 2.8 258.1 126.1 5,2'-O-dimethylcytidine 5.64 272.1 126.1 5-methyldihydrouridine 2.09 261.11 129.1 5-methyl-2-thiouridine 6.99 275.11 143.1 5-methyluridine 4.11 259.11 127.1 5,2'-O-dimethyluridine 7.54 273.11 127.1 N6,N6-dimethyladenosine 15.64 296.1 164.2 N6,N6,2'-O-trimethyladenosine 18.65 310.1 164.2 N6-methyladenosine 10.77 282.1 150.1 N6,2'-O-dimethyladenosine 13.17 296.1 150.1 O6-methylguanine 10.18 298.1 166.2 N6-methyl-N6-threonylcarbamoyladenosine_1 13.16 427.1 295.2 N6-methyl-N6-threonylcarbamoyladenosine_2 13.16 427.1 150.1 7-methylguanosine 3.55 298.1 166.2 8-methyladenosine 10.58 282.1 150.1 5-methoxycarbonylmethyl-2-thiouridine_1 9.16 333.11 201.1 5-methoxycarbonylmethyl-2-thiouridine_2 9.16 355.1 223.1 5-methoxycarbonylmethyluridine_1 6.08 317.11 185.2 5-methoxycarbonylmethyluridine_2 6.08 339.1 207.2 5-methoxycarbonylmethyl-2'-O-methyluridine 9.44 331.1 185.1 uridine 5-oxyacetic acid methyl ester_1 7.42 333.11 201.1 uridine 5-oxyacetic acid methyl ester_2 7.42 333.11 141.1 uridine 5-oxyacetic acid methyl ester_3 7.42 355.1 223.1 5-methylaminomethyl-2-thiouridine_1 2.95 304.1 172.1 5-methylaminomethyl-2-thiouridine_2 2.95 304.1 141 5-methoxyuridine 4.36 275.11 143.1 2-methylthio-N6-isopentenyladenosine_1 28.12 382.1 250.2 2-methylthio-N6-isopentenyladenosine_2 28.12 382.1 182.1 2-methylthio-N6-methyladenosine 20.25 328.1 196.1 5-carbamoylmethyluridine_1 1.67 302.1 170.1 5-carbamoylmethyluridine_2 1.67 302.1 125.1 pseudouridine 0.97 245.11 209.1 adenosine 6.9 268.1 136.1 cytidine 1.31 244.1 112.1 guanosine 4.12 284.1 152.1 inosine 3.84 291.1 159.1 uridine 1.92 245.11 113 2-thiocytidine 2.3 260.1 128.1 2-thiouridine 4.42 261.1 129.1 2-thio-2'-O-methyluridine 9.93 275.1 129 4-thiouridine 4.77 261.11 129.1 N6-threonylcarbamoyladenosine_1 10.36 413.11 136.1 N6-threonylcarbamoyladenosine_2 10.36 413.11 281.2 2'-O-methyluridine 5.03 259.11 113 METABOLITES_END #END