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Nat Methods. 2014 May;11(5):566-71. doi: 10.1038/nmeth.2886. Epub 2014 Mar 23.

Function-based identification of mammalian enhancers using site-specific integration.

Author information

1
Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
2
1] Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA. [2] Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, California, USA.
3
1] Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. [2] Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
4
1] Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA. [2] Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, California, USA. [3] Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA. [4] Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA.
5
1] Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. [2] US Department of Energy Joint Genome Institute, Walnut Creek, California, USA. [3] School of Natural Sciences, University of California, Merced, Merced, California, USA.
6
1] Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. [2] US Department of Energy Joint Genome Institute, Walnut Creek, California, USA.

Abstract

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

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PMID:
24658141
PMCID:
PMC4008384
DOI:
10.1038/nmeth.2886
[Indexed for MEDLINE]
Free PMC Article

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