Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing.
Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.|BloodCenter of Wisconsin, Blood Research Institute, 8733 West Watertown Plank Road, Milwaukee, WI, 53226, USA.
Steven
S
Blinka
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.|BloodCenter of Wisconsin, Blood Research Institute, 8733 West Watertown Plank Road, Milwaukee, WI, 53226, USA.
Michael H
MH
Reimer
BloodCenter of Wisconsin, Blood Research Institute, 8733 West Watertown Plank Road, Milwaukee, WI, 53226, USA.
Kirthi
K
Pulakanti
Department of Biostatistics and Computational Biology, Harvard TH Chan School of Public Health, Dana-Farber Cancer Institute, Boston, MA, USA.
Luca
L
Pinello
Department of Biostatistics and Computational Biology, Harvard TH Chan School of Public Health, Dana-Farber Cancer Institute, Boston, MA, USA.
Guo-Cheng
GC
Yuan
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA. sridhar.rao@bcw.edu.|BloodCenter of Wisconsin, Blood Research Institute, 8733 West Watertown Plank Road, Milwaukee, WI, 53226, USA. sridhar.rao@bcw.edu.|Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA. sridhar.rao@bcw.edu.
Sridhar
S
Rao
D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology; D011401:Promoter Regions, Genetic; D062085:RNA, Long Noncoding / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*; D014158:Transcription, Genetic
D062085:RNA, Long Noncoding
Chromatin immunoprecipitation sequencing; ENCODE; Global run on sequencing; Noncoding RNA; Transcribed enhancer; eRNA
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