Preface
References
Contents
Contributors
Chapter 1: Cellular Fractionation and Isolation of Chromatin-Associated RNA
1 Introduction
2 Materials
2.1 Cellular Fractionation and RNA Isolation
2.2 Agarose Gel Electrophoresis
3 Methods
3.1 Nuclei Isolation by Differential Centrifugation
3.2 Nuclear Fractionation
3.3 Gel Electrophoresis
4 Notes
References
Chapter 2: Knockdown of Nuclear-Located Enhancer RNAs and Long ncRNAs Using Locked Nucleic Acid GapmeRs
1 Introduction
2 Materials
3 Methods
3.1 Transfection for Suspension Cells
3.1.1 Cell Preparation
3.1.2 LNAs Transfection Mixture Preparation
3.2 “Reverse” Transfection for Adherent Cells
3.2.1 Cell Preparation
3.2.2 LNAs Solution Preparation
4 Notes
References
Chapter 3: Visualization of Enhancer-Derived Noncoding RNA
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.2.1 Preparation of Probe
2.2.2 Cell Culture
2.2.3 Fixation, Permeabilization, and Hybridization
2.2.4 Washing, TSA and Mounting of Cover Slips
3 Methods
3.1 Probe Design
3.2 Probe Labeling
3.3 Probe Purification
3.4 Cell Culture, Fixation, and Permeabilization
3.5 Hybridization
3.6 Washes
3.7 Tyramide Signal Amplification
3.8 Counterstaining and Mounting
3.9 Imaging and Image Processing
4 Notes
References
Chapter 4: UV-RNA Immunoprecipitation (UV-RIP) Protocol in Neurons
1 Introduction
2 Materials
2.1 Buffers
2.2 Tissue Culture
2.3 Other (See Note 1)
3 Methods
3.1 Cell Culture
3.2 UV Cross-linking
3.3 Cell Lysis and RNA Immunoprecipitation (See Note 3)
3.4 Elution
3.5 Detecting RNA (See Note 4)
4 Notes
References
Chapter 5: Mapping Long Noncoding RNA Chromatin Occupancy Using Capture Hybridization Analysis of RNA Targets (CHART)
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions
2.3 Reagents and Consumables
3 Methods
3.1 Preparation of Nuclei from Cross-Linked Cells
3.2 RNase H Mapping to Identify Regions of the Long ncRNA That Are Accessible for Hybridization
3.3 CHART Pull down
3.4 Nucleic Acid Purification to Test for Enrichment of Target Long ncRNA and Associated DNA Targets
4 Notes
References
Chapter 6: Detecting Long-Range Enhancer–Promoter Interactions by Quantitative Chromosome Conformation Capture
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment and Software
3 Methods
3.1 Experimental Design
3.2 Cross-Linking and Digestion
3.3 Ligation and Reverse Cross-Linking
3.4 DNA Purification
3.5 Analysis of 3C Library
3.5.1 Assessment of Digestion Efficiency
3.5.2 TaqMan qPCR Analysis of 3C Library
4 Notes
References
Chapter 7: Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET)
1 Introduction
2 Materials
2.1 Cell Culture
2.2 ChIP
2.3 Multiplex ChIA-PET
3 Methods
3.1 Cell Culture
3.2 ChIP
3.2.1 Single Cross-linking of Chromatin-Bound Proteins and Cell Harvesting (Suspension Cell)
3.2.2 Cell Lysis
3.2.3 Fragmentation of Chromatin
3.2.4 Washing and Preclearing of Chromatin
3.2.5 Coating of Antibody onto Magnetic Beads
3.2.6 Chromatin Immunoprecipitation
3.2.7 Washing and Elution of Immunoprecipitated DNA–Protein Complexes
3.3 Multiplex ChIA-PET
3.3.1 End Blunting of ChIP DNA Fragments
3.3.2 Ligation of Half-Linkers to Polished Ends (Refer to Subheading 5 for Sequence Information)
3.3.3 Addition of Phosphate Group to 5′-Ends of the Linkered DNA Fragments
3.3.4 Elution of Chromatin–DNA Complex
3.3.5 Circularization of Linkered DNA Fragments
3.3.6 Decross-linking of Chromatin–DNA Complex (Removal of Protein)
3.3.7 DNA Purification
3.3.8 MmeI Digestion to Release the Captured iPETs
3.3.9 Preparation of Dynabeads and Immobilization of iPET DNA
3.3.10 Ligation of Adaptors A and B to the Immobilized iPet-DNA
3.3.11 Nick Translation of Paired-End-Tag (PET) Constructs on Dynabeads
3.3.12 QC PCR Amplification for Viewing the iPETs
3.3.13 QC Gel Loading
3.3.14 Scale-Up PCR Amplification for Viewing the iPETs
3.3.15 Scale-Up Gel Loading
3.3.16 Gel Extraction Protocol
4 Notes
5 Appendix
References
Chapter 8: Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing
1 Introduction
2 Materials
2.1 Solutions
2.2 Lab Equipment
2.3 Chemicals
2.4 Kits
2.5 ENCODE Antibodies
3 Methods
3.1 Wet Lab Protocol for ChIP
3.1.1 Cell Preparation and Chromatin Immunoprecipitation
Prior to Day 1
Day 1
Day 2
Day 3
Day 4
3.1.2 ChIP-Seq Library Generation and Validation
3.2 Dry Bench Analysis of ChIP-Seq to Identify Putative Enhancers
3.2.1 Data Mining and Retrieval
Public ChIP-Seq Data Files
Private ChIP-Seq Data from Server
Quality checking FASTQ files
Mapping Reads to the Reference Genome
3.2.2 Peak Calling
3.2.3 Putative Enhancer Detection
3.3 Validating ChIP-Seq Data
3.3.1 Detection of Transcribed Enhancers by GRO-Seq Overlay
3.3.2 Wet Bench Approach to Validate Presence of H3K4me1, H3K27Ac, RNAPII, and Tissue Specific eRNAs
4 Notes
References
Chapter 9: Global Run-On Sequencing (GRO-Seq)
1 Introduction
2 Materials
2.1 Nuclei Isolation
2.2 Nuclear Run-On
2.3 RNA Immunoprecipitation
2.4 Library Preparation
3 Methods
3.1 Nuclei Isolation
3.2 Nuclear Run-On
3.3 RNA Immunoprecipitation
3.4 Library Preparation
4 Notes
References
Chapter 10: Computational Approaches for Mining GRO-Seq Data to Identify and Characterize Active Enhancers
1 Introduction
1.1 Transcriptional Enhancers Function as Genomic Regulatory Elements
1.2 Properties and Features of Active Enhancers
1.3 Identifying and Characterizing Enhancer Transcripts
1.4 Using GRO-Seq and Related Approaches to Identify and Study Active Enhancers
2 Materials: Computer, Data, and Software
3 Methods
3.1 Processing and Aligning GRO-Seq Data
3.1.1 Quality Control and Trimming the Adapter and polyA Sequences from the GRO-Seq Reads
3.1.2 Aligning the Trimmed GRO-Seq Reads to the Reference Genome
3.2 Analyzing GRO-Seq Data Using groHMM and Other Computational Tools
3.3 Identification of Active Enhancers from GRO-Seq Data
3.3.1 De Novo Identification of Enhancers Using GRO-Seq Data
3.3.2 Identification of Known TF Binding Sites That Are Actively Transcribed Using GRO-Seq Data
3.4 Associating Newly Identified Enhancers with TF Motifs
3.5 Associating Newly Identified Enhancers with Putative Target Genes
3.6 Identifying Cell Type-Specific Enhancers Using GRO-Seq Data
3.7 Integration with Other Genomic Data and Other Bioinformatic Analyses
3.8 Validation of Genomic Results Using Enhancer-Specific Molecular Biology Techniques
References
Chapter 11: Evaluating the Stability of mRNAs and Noncoding RNAs
1 Introduction
2 Materials
3 Methods
3.1 Cell plating
3.2 Actinomycin D Treatment
3.3 RNA Isolation (TRIzol Protocol)
3.4 Removal of Contaminating DNA
3.5 RNA Analysis
3.6 Transcript Quantification by Reverse Transcription Followed by Quantitative PCR (RT-qPCR)
3.7 Genome-Wide Transcript Quantification by DNA Oligoarray Hybridization
3.8 RNA Stability Analysis
4 Notes
References
Chapter 12: A Novel Method to Quantify RNA–Protein Interactions In Situ Using FMTRIP and Proximity Ligation
1 Introduction
2 Materials
2.1 Cell Culture and Reagents
2.2 FMTRIPs Assembly and Delivery
2.3 Proximity Ligation Assay
2.4 Microscope Operation and Processing
3 Methods
3.1 Neutravidin Labeling with Flag Peptides
3.2 Oligonucleotide Labeling with Fluorophores
3.3 FMTRIPs Assembly
3.4 Delivery to Cells
3.5 Proximity Ligation Assay
3.6 Detection and Analysis
3.7 Conclusions and Future Perspectives
4 Notes
References
Chapter 13: In Silico Promoter Recognition from deepCAGE Data
1 Introduction
1.1 The CAGE Techniques and the FANTOM Consortium
1.2 Methods for the Analysis of CAGE Data
2 Materials
3 Methods
3.1 The DPI Algorithm
3.2 Practical Usage of the DPI Software
3.3 The DPI Output
3.4 The PROmiRNA Software
3.5 Practical Usage of the PROmiRNA Software
3.6 The PROmiRNA Output
3.7 Case Study: Prediction of miRNA Promoters in Gm12878
3.7.1 Application of PROmiRNA to Gm12878 CAGE Data
3.7.2 Application of DPI to Gm12878 CAGE Data
4 Notes
References
Chapter 14: Bioinformatics Pipeline for Transcriptome Sequencing Analysis
1 Introduction
1.1 RNA-Seq Technology
1.2 The STAR: Cufflinks: RSEM pipeline
1.3 Alternative Pipelines
2 Materials
2.1 RNA-Seq FASTQ Reads
2.2 Reference Genome and Annotation Files
2.3 Software Installation
2.4 SAMtools
2.5 STAR
2.6 Cufflinks
2.7 RSEM
3 Methods
3.1 Mapping
3.1.1 Making the STAR Indices
3.1.2 Input Files and Arguments
3.1.3 Command
3.1.4 Output Files
3.1.5 Mapping the Reads
3.1.6 Input Files and Arguments
3.1.7 Command
3.1.8 Output Files
3.2 Transcriptome Reconstruction/Assembly
3.2.1 Cufflinks
3.2.2 Input Files and Arguments
3.2.3 Command
3.2.4 Output File
3.3 Transcript and Gene Quantifications
3.3.1 Input Files and Arguments
3.3.2 Command
3.3.3 Output Files
3.4 Running the Quantification Process
3.4.1 Input Files and Arguments
3.4.2 Command
3.4.3 Output Files
4 Notes
References
Chapter 15: CRISPR/Cas9 Genome Editing in Embryonic Stem Cells
1 Introduction
2 Material
2.1 Vector and Guide RNA
2.2 Transfection and Selection of Murine Embryonic Stem Cells
2.3 ES Cell Clone Lysis and Screening
2.4 Clone Expansion
3 Methods
3.1 Producing the CRISPR Vector
3.2 ESC Culture
3.3 ES Cell Clone Lysis and Genotyping
3.4 Clone Expansion
4 Notes
References
Chapter 16: Targeted Gene Activation Using RNA-Guided Nucleases
1 Introduction
2 Materials
2.1 Construction of sgRNA Expression Plasmids
2.2 Activation of Target Gene Expression
2.3 Analysis of mRNA Expression
3 Methods
3.1 Design and Construction of sgRNA Expression Plasmids
3.2 Activation of Target Gene Expression in Mammalian Cells
3.3 Analysis of Gene Expression by qPCR
4 Notes
References
ERRATUM TO
Index