Circular RNAs: Methods and Protocols

Preface......Page 5
 References......Page 6
Contents......Page 8
Contributors......Page 10
 1 Introduction......Page 13
  2.1 Hardware and Environment......Page 14
  2.3 Data Requirements......Page 15
  3.1 Running the CIRI Pipeline Step by Step on the Test Dataset......Page 16
  3.2 An Automated Pipeline for Detecting and Reconstructing circRNAs......Page 18
  3.3 Output of the CIRI-Full Pipeline......Page 19
 Reference......Page 20
 1 Introduction......Page 21
  2.2 Required Software Environment......Page 22
  3.1 Quality Control of Obtained Sequencing Data with FastQC......Page 23
  3.3 Removal of Remaining Ribosomal RNA from Sequencing Data......Page 24
  3.4 Principal Read Mapping with STAR......Page 25
  3.5 CircRNA Detection with DCC......Page 27
  3.6 Test for Host: Independently Regulated circRNAs with CircTest......Page 30
  3.7 Full Circle Characterization with FUCHS......Page 31
  4.1 DCC and CircTest......Page 33
  4.2 Fuchs......Page 34
 References......Page 36
 1 Introduction......Page 38
  3.1 Generate circRNA Junction Sequences......Page 40
  3.2 Align RNA-seq Reads to the circRNA Junction Scaffold......Page 44
  3.3 Count Reads that Align to circRNA Junctions Using Featurecounts......Page 46
  3.4 Normalize circRNA Counts to Compare circRNA Expression Across Conditions......Page 47
 4 Notes......Page 49
 References......Page 50
 1 Introduction......Page 53
 2 Materials......Page 54
  3.2 CircRNA Genomic Sequence and Mature circRNA Sequence (Fig. 3)......Page 55
  3.3 Specific circRNA Gene (Fig. 4)......Page 58
  3.5 miRNAs Targeting circRNAs (Fig. 6)......Page 59
  3.6 CircRNA Divergent Primer Design (Fig. 7)......Page 61
 4 Summary......Page 63
 References......Page 66
 1 Introduction......Page 67
  2.1 Primer Design......Page 68
  3.1 Primer Design......Page 69
  3.2 PCR Semiquantitative PCR......Page 70
  3.3 Quantitative PCR......Page 72
 4 Notes......Page 73
 References......Page 77
 1 Introduction......Page 78
 2 Materials......Page 79
  3.3 Coverslip Preparation......Page 80
  3.7 Hybridization......Page 81
  3.9 Imaging and High-Precision Localizations......Page 82
 4 Notes......Page 83
 References......Page 84
 1 Introduction......Page 85
  2.1 Equipment......Page 91
  2.4 Fixation......Page 92
  2.6 Washing, DAPI Staining, and Mounting in Oxygen Removal Medium (Day 2)......Page 93
  3.1 Design and Preparation of Probes......Page 94
  3.3 Hybridize RNAs (Day 1)......Page 95
  3.5 Mounting in Oxygen Removal Buffer (Day 2)......Page 96
  3.6 Imaging (Day 2)......Page 97
  3.8 RNA Spot Quantification......Page 98
 4 Notes......Page 99
 References......Page 102
 1 Introduction......Page 105
  2.1 Construction of the circR Cloning Vector......Page 106
  2.2 Tissue Culture and Transfection......Page 107
  2.3 qPCR Validation of circRNA Expression......Page 108
   3.1.2 The Downstream Intron Cloning (the Inverted Upstream Intron Sequence) (Note 2)......Page 109
  3.2 Inserting Desired Circular RNA Sequence (Includes the Endogenous Flanking Genomic Sequence ~200 bp Upstream and Downstream, Respectively)......Page 110
  3.4 qPCR Validation of circRNA Expression......Page 111
 4 Notes......Page 112
 References......Page 113
 1 Introduction......Page 114
  2.1 Plasmid Construction......Page 115
  2.4 SDS Polyacrylamide Gel......Page 116
  2.5 Immunoblotting and Flow Cytometry......Page 117
  3.1 Plasmid Construction......Page 118
  3.2 Cell Culture and Transfection......Page 119
  3.3 Reverse Transcription and PCR......Page 120
  3.4 Western Blot......Page 121
  3.5 Flow Cytometry......Page 123
 4 Notes......Page 124
 References......Page 125
 1 Introduction......Page 126
  2.2 Denaturing Polyacrylamide Gel......Page 128
  2.4 DIG-­Detection System......Page 129
  3.1 Agarose Gel Electrophoresis of RNA......Page 130
  3.2 Denaturing Polyacrylamide Gel Electrophoresis of RNA......Page 131
  3.4 Northern Probe Design......Page 132
  3.5 Detection of Specific circRNAs by DIG-labeled Probes......Page 134
  3.6 Detection of Specific circRNAs by Radiolabeled Oligonucleotides......Page 136
  3.8 Analysis of circRNAs by RNase H Cleavage......Page 137
 4 Notes......Page 138
 References......Page 140
 1 Introduction......Page 141
 2 Materials......Page 142
  3.3 Nonradioactive Probe Preparation......Page 144
  3.5 Detection......Page 145
 4 Notes......Page 146
 References......Page 147
 1 Introduction......Page 148
  2.1 circRNA Biochemistry......Page 149
   2.2.3 PAGE (Polyacrylamide Gel Electrophoresis)......Page 151
  3.1 General Considerations......Page 152
  3.3 RNase H Digest......Page 153
  3.4 Alkaline Hydrolysis......Page 154
  3.6 Agarose Gel Electrophoresis......Page 155
  3.7 PAGE......Page 156
   3.9.2 Hybridize Membrane......Page 158
 4 Notes......Page 159
 References......Page 162
 1 Introduction......Page 163
 2 Materials......Page 166
  3.4 Sucrose Gradient Fractionation......Page 167
 4 Notes......Page 168
 References......Page 170
 1 Introduction......Page 171
  2.2 Apparatus......Page 175
  2.4 Deprotection......Page 176
  2.11 In Vitro Transcription......Page 177
  2.16 Circularization of Single-Stranded or Partially Single-Stranded RNAs......Page 178
  3.1 Chemical RNA Synthesis and Deprotection......Page 179
   3.3.1 RNA Precipitation Using Ethanol After Chemical Synthesis......Page 180
  3.4 In Vitro Transcription and Product Purification......Page 181
  3.5 In Vitro Transcription with GMP Priming and Product Purification......Page 182
  3.9 RNA Circularization with the Ligation Junction Positioned in a Double-Stranded Region......Page 183
 References......Page 184
 1 Introduction......Page 185
  2.1 Denaturing Polyacrylamide Gel Electrophoresis (PAGE) of RNA......Page 186
  2.2 Chemical Synthesis of linear 5′-Phosphorylated RNA......Page 189
  3.1 Preparation of Denaturing Polyacrylamide Gel......Page 190
  3.3 Chemical Synthesis of linear 5′-Phosphorylated RNA (Fig. 3, See Note 4)......Page 191
  3.5 Synthesis of Circular RNA by Ligation (Figs. 2, 3d, and 4b)......Page 193
  3.6 Circularity Check of RNA Using RNase R (Fig. 5, See Note 12)......Page 194
 4 Notes......Page 195
 References......Page 196
 1 Introduction......Page 197
   2.2.4 clipSearch Software......Page 198
  3.1 Discovering circRNAs from RNA-Seq Sequencing Data......Page 200
  3.2 Annotating circRNAs Using circAnno Software......Page 202
  3.3 Predicting AGO-Binding Sites from CLIP-Seq Data......Page 203
  3.4 Identifying the circRNA-miRNA Interactions Using clipSearch Software......Page 205
  3.5 Browsing circRNA-miRNA Interactions Predicted by ClipSearch......Page 207
  3.6 Comparative Analysis of circRNA-miRNA Interactions Using starBase Genome Browser......Page 208
 4 Notes......Page 209
 References......Page 210
 1 Introduction......Page 212
   2.2.1 Vector Construction......Page 213
   2.2.2 Cell Culture......Page 214
   3.1.3 Immunoprecipi­tation of Protein-RNA Complexes......Page 216
   3.1.5 Quantitative Real-Time PCR Analysis......Page 217
   3.2.2 Transfection......Page 218
 4 Notes......Page 219
 References......Page 221
Index......Page 222