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|a 0128235861
|q (electronic bk.)
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|a 9780128235867
|q (electronic bk.)
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|a (OCoLC)1268546688
|z (OCoLC)1268206212
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|a RNA modification enzymes /
|c edited by Jane E. Jackman.
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|a Cambridge, MA :
|b Academic Press,
|c 2021.
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|a 1 online resource (1 volume) :
|b illustrations (chiefly color)
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|a text
|b txt
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|a computer
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|a online resource
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|a Methods in enzymology ;
|v volume 658
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|a Print version record.
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|a Intro -- RNA Modification Enzymes -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis -- 1. Introduction -- 2. Characterization of chemical modifications in RNA -- 2.1. Identity and census of resident modifications -- 2.2. Locating the chemical modification in the RNA sequence -- 3. Tools to characterize modified RNA sequence -- 3.1. Equipment (available through multiple vendors) -- 3.2. Chemicals -- 4. Protocols -- 4.1. RNA hydrolysis to nucleosides -- 4.2. LC-MS based identification of modifications -- 4.3. Oligonucleotide generation for location-specific information -- 4.3.1. Nucleobase-specific ribonucleases -- 4.3.2. Nucleoside-preferential ribonucleases (alternate approach) -- 4.3.3. Non-specific ribonucleases -- 4.4. LC-MS analysis for sequencing the oligonucleotides -- 4.5. Data analysis -- 5. Summary -- Acknowledgment -- References -- Chapter Two: Mapping of 7-methylguanosine (mC) RNA modifications by AlkAniline-Seq -- 1. Introduction -- 2. Chemical approaches for mapping of mC -- 2.1. Cleavage of N-glycosidic bond and/or detection of the reverse transcription arrest -- 2.2. Selective ligation to the 5-phosphate resulting from RNA abasic site -- 3. Overview of AlkAniline-Seq protocol -- 3.1. RNA fragmentation step -- 3.2. De-phosphorylation -- 3.3. Aniline cleavage -- 3.4. Adapter ligation and barcoding -- 3.5. Sequencing -- 3.6. Data analysis -- 4. Analysis of tRNA and rRNA in total RNA fraction -- 4.1. Equipment -- 4.2. Chemicals -- 4.3. Consumables and kits for library preparation and sequencing -- 4.4. Biological material -- 5. AlkAniline-Seq protocol -- 5.1. RNA fragmentation in bicarbonate buffer at 95C -- 5.2. De-phosphorylation by Antarctic phosphatase -- 5.3. Aniline cleavage.
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|a 5.4. Library preparation using NEBNext multiplex small RNA library prep set for Illumina -- 5.5. Purification of the library using GeneJET PCR purification kit -- 5.6. Library quantification and quality assessment -- 5.7. Sequencing of the library -- 5.8. Bioinformatic analysis -- 6. Data analysis and interpretation -- 7. Limitations -- 8. Summary -- Acknowledgments -- References -- Chapter Three: Facile detection of RNA phospho-methylation in cells and tissues -- 1. Introduction -- 1.1. Before you begin -- 1.1.1. Buffer preparation -- 1.1.2. Key resources table -- 1.2. Materials and equipment -- 1.2.1. Cell culture -- 1.2.1.1. Equipment -- 1.2.1.2. Reagents -- 1.2.2. Protein and RNA extraction -- 1.2.2.1. Equipment -- 1.2.2.2. Reagents -- 1.2.3. Antarctic phosphatase or terminator treatment -- 1.2.3.1. Equipment -- 1.2.3.2. Reagents -- 1.2.4. RNA cleanup -- 1.2.5. Northern blot -- 1.2.5.1. Equipment -- 1.2.5.2. Reagents -- 1.2.5.3. Alternatives -- 2. Protocol -- 2.1. Seeding cells -- 2.2. Preparing protein and RNA extracts from same cells with the Norgen RNA/Protein Plus kit (Product #48200) -- 2.3. AP-shift assay for tRNA -- 2.4. Terminator assay -- 2.5. RNA clean-up using the Qiagen RNeasy MinElute kit -- 2.6. Northern blotting -- 3. Expected outcomes -- 4. Quantification and statistical analysis -- 5. Advantages -- 6. Limitations -- 7. Optimization and troubleshooting -- 7.1. Problem -- 7.2. Potential solution to optimize the procedure -- 8. Safety considerations and standards -- 9. Alternative methods/procedures -- References -- Chapter Four: Quantitative probing of glycosylated queuosine modifications in tRNA -- 1. Introduction -- 2. Methods -- 2.1. Total RNA deacylation -- 2.2. Acid denaturing polyacrylamide gel electrophoresis -- 2.3. Nonradioactive Northern blot quantification -- 3. Notes -- Acknowledgments -- References.
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|a Chapter Five: CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei -- 1. Introduction -- 2. Protein affinity purification and in vivo proximity labeling -- 2.1. In situ CTS-tagging -- 2.2. Purification of protein and ribonucleoprotein complexes -- 2.2.1. Equipment -- 2.2.2. Buffers and reagents -- 2.2.3. Procedure -- 2.3. In vivo proximity biotinylation -- 2.3.1. Equipment -- 2.3.2. Buffers and reagents -- 2.3.3. Procedure -- 3. UV-crosslinking tandem affinity purification sequencing (CTAP-SEQ) -- 3.1. UV-crosslinking, purification and RNA-Seq library preparation -- 3.1.1. Equipment -- 3.1.2. Buffers and reagents -- 3.1.3. Procedure -- 3.2. CTAP-SEQ data analysis -- 3.2.1. Data pre-processing -- 3.2.1.1. Adaptor trimming -- 3.2.1.2. Handling unique molecular identifier (UMI) -- 3.2.1.3. Filtering out nuclear genome-encoded RNAs -- 3.2.2. Binding sites distribution along RNAs of interest -- 4. Immunofluorescence imaging of CTS-tagged proteins -- 4.1. Equipment -- 4.2. Reagents and buffers -- 4.3. Procedure -- 5. Summary -- Acknowledgments -- References -- Chapter Six: Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry -- 1. Introduction -- 2. Materials -- 2.1. Cell culture -- 2.2. RNA isolation -- 2.3. RNA purification -- 2.4. PCR and in vitro transcription -- 2.5. AlkB in vitro assay -- 2.6. Primers, oligonucleotides and templates -- 2.7. Digestion -- 2.8. LC-MS -- 3. Methods -- 3.1. General workflow of an oligonucleotide (ON)-MS experiment -- 3.1.1. Considerations for biological samples (RNA substrates) -- 3.1.2. Considerations for RNA digestion -- 3.1.3. Considerations for mass spectrometry -- 3.2. RNA substrates -- 3.2.1. Cell culture -- 3.2.2. T7 in vitro transcribed RNA as substrate or internal standard -- 3.2.3. Synthetic oligonucleotides.
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|a 3.3. Processing of RNA -- 3.3.1. RNA isolation for native RNA -- 3.3.2. SEC separation for native tRNA and in vitro transcribed tRNA -- 3.3.3. RNA in vitro demethylation with AlkB -- 3.4. RNase T1 digest for ON-MS -- 3.5. Low- and high-resolution mass spectrometry -- 3.5.1. Low-resolution QQQ for oligonucleotide mass spectrometry -- 3.5.2. Comparison of chromatographic set-up for nucleoside-MS and ON-MS -- 3.5.3. High-resolution orbitrap for ON-MS -- 3.6. Data analysis -- 3.6.1. Agilent�s Qualsoftware -- 3.6.2. In silico tools and data analysis -- 4. Notes -- Acknowledgments -- References -- Chapter Seven: RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes -- 1. Introduction -- 2. Factors to consider when designing a RIP assay -- 3. Step-by-step method details -- 3.1. Materials and equipment -- 4. Troubleshooting -- 5. Summary -- Acknowledgment -- Reference -- Chapter Eight: Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites -- 1. Before you begin -- 2. Key resources table -- 3. Materials and equipment -- 3.1. Equipment -- 3.2. Materials -- 3.3. Reagents -- 3.3.1. Alternatives -- 4. Step-by-step method details -- 4.1. Recombinant expression and purification of METTL3-METTL14 -- 4.1.1. Generation of recombinant bacmids in E. coli DH10Bac cells -- 4.1.2. Cultivation and transfection of Sf21 insect cells -- 4.1.3. Preparation of V1 baculovirus stock -- 4.1.4. SDS-PAGE analysis -- 4.1.5. Small-scale expression test -- 4.1.6. Large-scale expression -- 4.1.7. Purification of METTL3-METTL14 -- 4.2. Synthesis of 5-[(R/S)-[(3S)-3-amino-3-carboxy-propyl]prop-2-yn-1-ylselenio]-5-deoxyadenosine (SeAdoYn) -- 4.2.1. In vitro MTase assay -- 4.3. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) -- 4.3.1. Cleanup via microspin G-25 columns -- 4.3.2. Enrichment via magnetic beads.
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|a 4.3.3. Detection via primer extension assay -- 4.4. PAGE -- 5. Expected outcomes -- 6. Quantification and data analysis -- 7. Advantages -- 8. Limitations -- 9. Optimization and troubleshooting -- 9.1. Problematic step: Generation of recombinant bacmids in E. coli DH10Bac cells -- 9.2. Problematic step: Synthesis of SeAdoYn -- 9.3. Problematic step: CuAAC -- 10. Safety considerations and standards -- 11. Alternative methods/procedures -- 11.1. Metabolic labeling -- 11.2. Extraction of total RNA from HeLa cells -- 11.3. Detection via fluorescence -- 11.4. End-point kinetics measurements -- Acknowledgments -- References -- Chapter Nine: Analysis of codon-specific translation by ribosome profiling -- 1. Introduction -- 2. Before you begin -- 2.1. Key resources table -- 2.2. Abbreviations -- 3. Step-by-step method details -- 3.1. Sample harvesting and RNase I digestion -- 3.1.1. HEK293T cells: Cell harvesting and extract preparation (Video 1) -- 3.1.2. HEK293T cells: RNA digestion -- 3.1.3. Yeast: Cell harvesting (Video 2) -- 3.1.4. Yeast: cell lysis (Video 3) -- 3.1.5. Yeast: Cell extract preparation and RNA digestion (Video 4) -- 3.2. Gradient ultracentrifugation and fractionation (Video 5) -- 3.3. RNA isolation from monosome fraction (Video 6) -- 3.4. Size selection of ribosome-protected fragments (RPF) by gel electrophoresis (Video 7) -- 3.5. Dephosphorylation (Video 8) -- 3.6. Adapter ligation -- 3.7. Reverse transcription (Video 9) -- 3.8. cDNA circularization (Video 10) -- 3.9. Test PCR (Video 11) -- 3.10. Library PCR -- 4. Expected outcomes -- 5. Quantification and statistical analysis -- 5.1. Data processing and quality control -- 5.2. Differential translation analysis -- 6. Advantages -- 7. Limitations -- 8. Optimization and troubleshooting -- 9. Safety considerations and standards -- 10. Alternative methods/procedures -- References.
|
| 650 |
|
0 |
|a Catalytic RNA.
|
| 650 |
|
0 |
|a Nucleotides.
|
| 650 |
|
0 |
|a Transfer RNA.
|
| 650 |
1 |
2 |
|a RNA Editing
|0 (DNLM)D017393
|
| 650 |
2 |
2 |
|a Nucleotides
|0 (DNLM)D009711
|
| 650 |
2 |
2 |
|a RNA, Transfer
|0 (DNLM)D012343
|
| 650 |
|
6 |
|a Ribozymes.
|0 (CaQQLa)201-0224271
|
| 650 |
|
6 |
|a Nucl�eotides.
|0 (CaQQLa)201-0012930
|
| 650 |
|
6 |
|a ARN de transfert.
|0 (CaQQLa)201-0029738
|
| 650 |
|
7 |
|a Catalytic RNA
|2 fast
|0 (OCoLC)fst00848887
|
| 655 |
|
4 |
|a Internet Resources.
|
| 655 |
|
4 |
|a Index not Present.
|
| 700 |
1 |
|
|a Jackman, Jane E.,
|e editor.
|
| 776 |
0 |
8 |
|i Print version:
|t RNA MODIFICATION ENZYMES.
|d [Place of publication not identified] : ELSEVIER ACADEMIC PRESS, 2021
|z 0128235853
|w (OCoLC)1241730447
|
| 830 |
|
0 |
|a Methods in enzymology ;
|v v. 658.
|
| 856 |
4 |
0 |
|u https://sciencedirect.uam.elogim.com/science/bookseries/00766879/658
|z Texto completo
|
