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Development. 2016 Nov 15;143(22):4272-4278. Epub 2016 Oct 5.

iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins.

Author information

1
Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg D-69120, Germany.
2
Deep Sequencing Core Facility, Cell Networks, University of Heidelberg, Im Neuenheimer 267, Heidelberg D-69120, Germany.
3
Developmental Genetics of the Nervous System, Max Planck Institute for Medical Research, Jahnstrasse 29, Heidelberg D-69120, Germany.
4
Focus Program Translational Neuroscience, University Medical Center, Johannes Gutenberg University Mainz, Langenbeckstr. 1, Mainz D-55131, Germany.
5
Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg D-69120, Germany jochen.wittbrodt@cos.uni-heidelberg.de.

Abstract

DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.

KEYWORDS:

Chromatin profiling; DamID; Epigenetics; Transcription factor; Transcriptional regulation

PMID:
27707796
PMCID:
PMC5117216
DOI:
10.1242/dev.139261
[Indexed for MEDLINE]
Free PMC Article

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