UID:
almafu_9958131247502883
Format:
1 online resource (595 p.)
Edition:
First edition.
ISBN:
0-12-801335-4
Series Statement:
Methods in Enzymology ; Volume 549
Content:
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in riboswitch discovery and validation, synthesis and sample prep methods for large RNAs, riboswitch structure and function methods, folding pathways and dynamics, and ligand interactions and thermodynamics.Continues the legacy of this premier serial with quality chapters authored by leaders in the fieldCovers research methods in biomineralization scienceContains sections on such topics as riboswitch discovery and valid
Note:
Description based upon print version of record.
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Front Cover; Riboswitch Discovery, Structure and Function; Copyright; Contents; Contributors; Preface; Volume 1; Volume 2; Part I: Riboswitch Discovery; Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites; 1. Introduction; 2. RNA Isolation and mRNA Enrichment; 2.1. Equipment and materials; 2.2. RNA isolation and quality control; 2.3. mRNA enrichment and quality control; 3. Genome-Wide Mapping of Transcription Start Sites by dRNA-Seq; 3.1. Equipment and materials; 3.2. Hydrolysis of triphosphate groups at mRNA 5-ends by TAP
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3.3. Ligation of adapter on 5-end of mRNAs3.4. cDNA first strand synthesis by random priming; 3.5. cDNA sizing on agarose gels; 3.6. PCR amplification; 3.7. Purification of the PCR products on Agencourt AMPure beads; 3.8. Quality control of the libraries; 3.9. Data analysis; 4. Genome-Wide Analysis of Transcript Length by RNA-Seq; 4.1. Strand-specific RNA-Seq library construction; 4.2. Nonoriented whole-transcript RNA-Seq library preparation; 5. Processing and Analysis of dRNA-Seq and RNA-Seq Data; 5.1. Softwares and supplementary files required for the analysis; 5.2. Protocol
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5.2.1. Trimming of the adapter sequences5.2.2. Creation of a reference index for bowtie 1; 5.2.3. Alignment of the reads to the reference genome; 5.2.4. Conversion of the SAM file to a sorted BAM file and creation of an index; 5.2.5. Visualization of the alignments on a genome browser; 6. Characterization of New Potential Riboswitches Using dRNA-Seq and RNA-Seq Analyses; References; Chapter Two: Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection; 1. Introduction; 2. Precautions; 3. Generating a Human Genomic DNA Pool; 3.1. Materials
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3.1.1. High molecular weight human genomic DNA3.1.2. Adapter oligonucleotide sequences; 3.1.3. Enzymes; 3.1.4. Buffers; 3.1.4.1. Tris/borate/EDTA buffer (10x); 3.1.5. Instruments and miscellaneous; 3.2. Procedures; 3.2.1. Preparation of genomic DNA; 3.2.2. Repairing genomic DNA ends; 3.2.3. Addition of 5 phosphate group onto genomic DNA; 3.2.4. Addition of 3 dA overhangs; 3.2.5. Adapter ligation; 3.2.6. PCR amplification; 4. In Vitro Selection of RNA Aptamers; 4.1. Materials; 4.1.1. Selection buffers; 4.1.2. Polyacrylamide gel electrophoresis; 4.1.3. Agarose gel electrophoresis
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4.1.4. Transcription4.1.5. Reverse transcription; 4.1.6. Polymerase chain reaction; 4.1.7. Enzymes; 4.1.8. Affinity column for in vitro selection; 4.2. Procedure; 4.2.1. Transcription; 4.2.2. Purification of transcribed product; 4.2.3. In vitro selection of RNA aptamers; 4.2.4. Reverse transcription of selected RNAs; 4.2.5. Polymerase chain reaction; 5. Structure-Based Searches for Naturally Occurring Aptamers; 5.1. Materials; 5.1.1. Unix compliant operating system; 5.1.2. RNABOB; 5.1.3. RNArobo; 5.2. Procedures; 5.2.1. Descriptor; 5.2.2. Sequence data; References
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Part II: Synthesis and Sample Prep Methods for Large RNAs
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English
Additional Edition:
ISBN 0-12-801122-X
Language:
English
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