Abstract
BackgroundChIP-seq is the method of choice for genome-wide studies of protein–DNA interactions. We describe a new method for ChIP-seq sample preparation, termed lobChIP, where the library reactions are performed on cross-linked ChIP fragments captured on beads.ResultsThe lobChIP method was found both to reduce time and cost and to simplify the processing of many samples in parallel. lobChIP has an early incorporation of barcoded sequencing adaptors that minimizes the risk of sample cross-contamination and can lead to reduced amount of adaptor dimers in the sequencing libraries, while allowing for direct decross-linking and amplification of the sample.ConclusionsWith results for histone modifications and transcription factors, we show that lobChIP performs equal to or better than standard protocols and that it makes it possible to go from cells to sequencing ready libraries within a single day.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0017-5) contains supplementary material, which is available to authorized users.
Highlights
chromatin immunoprecipitation (ChIP)-seq is the method of choice for genome-wide studies of protein–deoxyribonucleic acid (DNA) interactions
Results library-on-beads ChIP-seq protocol (lobChIP) permits a shorter ChIP‐seq workflow which results in high concordance with public datasets We reasoned that when the desired end product of a ChIP experiment is a sequencing library, it would be advantageous to perform library reactions during the ChIP step rather than after DNA purification
We performed ChIP using standard protocols until the IP washes and end repair, A-tailing and ligation reactions were done directly on cross-linked chromatin attached to magnetic beads (Figure 1a), with brief washes with phosphate-buffered saline (PBS) in between the reactions to remove enzymes
Summary
ChIP-seq is the method of choice for genome-wide studies of protein–DNA interactions. The ability to decipher the regulatory information in the genome and epigenome is essential for understanding how transcription is controlled and how genetic variation affects disease states. Transcription factors (TF) bind DNA in a sequence-specific manner and can have either a repressive or activating function, and recent work shows that sequence variants affecting TF binding sites are the underlying mechanism of sequence-specific gene regulation [1] that in turn can lead to altered histone modification states. To get a better understanding of the complex regulatory networks in a cell, many transcription factors and histone modifications need to be analyzed together under different conditions. Since the introduction of massively parallel next-generation sequencers, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the method of choice for genome-wide detection of regulatory elements [2].
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