Abstract
Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. Current barcoding strategies aim to improve assay throughput and scalability but intense sample handling and lack of standardization over cell types, cell numbers and epitopes hinder wide-spread use in the field. Here, we present a barcoding method to enable high-throughput ChIP-seq using common molecular biology techniques. The method, called RELACS (restriction enzyme-based labeling of chromatin in situ) relies on standardized nuclei extraction from any source and employs chromatin cutting and barcoding within intact nuclei. Barcoded nuclei are pooled and processed within the same ChIP reaction, for maximal comparability and workload reduction. The innovative barcoding concept is particularly user-friendly and suitable for implementation to standardized large-scale clinical studies and scarce samples. Aiming to maximize universality and scalability, RELACS can generate ChIP-seq libraries for transcription factors and histone modifications from hundreds of samples within three days.
Highlights
Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins
Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is a key technique for exploring the genomic location of bound proteins and histone modifications, which has significantly contributed to our understanding of gene regulation and epigenetic changes in healthy and diseased cells
The key of the protocol is to work with intact nuclei that can be precipitated to efficiently wash and remove the different master mixes during the chromatin fragmentation and barcoding process and to reduce volumes after pooling of many barcoded samples
Summary
Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. Recent years have seen a number of methodological advances[1] to reduce the input requirements for ChIP-seq from millions to few thousands of cells per assay, by using improved sample preparation[2,3,4,5,6], more sensitive library preparation[4,7,8], and microfluidic devices[9,10] Despite these improvements, ChIP-seq still suffers from limitations imposed by the complexity of the protocol, lack of standardization across cell types and epitopes, technical variability, low efficiency for proteins weakly bound to chromatin, and limited throughput. Barcode sequences ligated to each starting sample are used to identify signals from each cell population during data analysis
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