Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.     

Perm-seq: Mapping Protein-DNA Interactions in Segmental Duplication and Highly Repetitive Regions of Genomes with Prior-Enhanced Read Mapping

Segmental duplications and other highly repetitive regions of genomes contribute significantly to cells’ regulatory programs. Advancements in next generation sequencing enabled genome-wide profiling of protein-DNA interactions by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). However, interactions in highly repetitive regions of genomes have proven difficult to map since short reads of 50–100 base pairs (bps) from these regions map to multiple locations in reference genomes. Standard analytical methods discard such multi-mapping reads and the few that can accommodate them are prone to large false positive and negative rates. We developed Perm-seq, a prior-enhanced read allocation method for ChIP-seq experiments, that can allocate multi-mapping reads in highly repetitive regions of the genomes with high accuracy. We comprehensively evaluated Perm-seq, and found that our prior-enhanced approach significantly improves multi-read allocation accuracy over approaches that do not utilize additional data types. The statistical formalism underlying our approach facilitates supervising of multi-read allocation with a variety of data sources including histone ChIP-seq. We applied Perm-seq to 64 ENCODE ChIP-seq datasets from GM12878 and K562 cells and identified many novel protein-DNA interactions in segmental duplication regions. Our analysis reveals that although the protein-DNA interactions sites are evolutionarily less conserved in repetitive regions, they share the overall sequence characteristics of the protein-DNA interactions in non-repetitive regions.     

jMOSAiCS: joint analysis of multiple ChIP-seq datasets

The ChIP-seq technique enables genome-wide mapping of in vivo protein-DNA interactions and chromatin states. Current analytical approaches for ChIP-seq analysis are largely geared towards single-sample investigations, and have limited applicability in comparative settings that aim to identify combinatorial patterns of enrichment across multiple datasets. We describe a novel probabilistic method, jMOSAiCS, for jointly analyzing multiple ChIP-seq datasets. We demonstrate its usefulness with a wide range of data-driven computational experiments and with a case study of histone modifications on GATA1-occupied segments during erythroid differentiation. jMOSAiCS is open source software and can be downloaded from Bioconductor [1].     

Das methodische Potenzial der neuen Sequenziertechnologien jenseits der Mutationssuche

In this article, a number of important applications for next generation sequencing (NGS)-based techniques are presented. For each method the technical principles are introduced, the application options in human genetic research using selected examples are illustrated and possible sources for detailed protocols are indicated. The following methods are described: RNA sequencing using NGS (RNA-seq), chromatin immunoprecipitation in combination with NGS (ChIP-seq), ribosome profiling, methylated DNA immunoprecipitation in combination with NGS (MeDIP-seq) and the HiC technique, an extension of the chromosome confirmation capture (3c) method.     

MM-ChIP enables integrative analysis of cross-platform and between-laboratory ChIP-chip or ChIP-seq data

The ChIP-chip and ChIP-seq techniques enable genome-wide mapping of in vivo protein-DNA interactions and chromatin states. The cross-platform and between-laboratory variation poses a challenge to the comparison and integration of results from different ChIP experiments. We describe a novel method, MM-ChIP, which integrates information from cross-platform and between-laboratory ChIP-chip or ChIP-seq datasets. It improves both the sensitivity and the specificity of detecting ChIP-enriched regions, and is a useful meta-analysis tool for driving discoveries from multiple data sources.     

染色质免疫沉淀技术分析HePG2细胞TCF7L2蛋白与IDE基因转录启动子的结合

TCF7L2是一种重要的转录因子,通过Wnt信号途径,调节葡萄糖代谢.胰岛素降解酶(IDE)是细胞水平催化胰岛素降解的最关键的酶,与2型糖尿病(T2DM)高血糖、胰岛素抵抗、高胰岛素血症密切相关.为了检测HePG2细胞内转录因子TCF7L2与IDE基因启动子区的结合情况,采用染色质免疫沉淀技术结合PCR技术检测IDE基因启动子序列.结果表明,在特异性TCF7L2抗体免疫沉淀的DNA片段中扩增出IDE基因启动子序列,因此证实在HePG2细胞内,TCF7L2蛋白可与IDE基因转录启动子的特异区域结合,进而可能参与IDE基因的表达调控.     

hmChIP: a database and web server for exploring publicly available human and mouse ChIP-seq and ChIP-chip data

hmChIP is a database of genome-wide chromatin immunoprecipitation (ChIP) data in human and mouse. Currently, the database contains 2016 samples from 492 ChIP-seq and ChIP-chip experiments, representing a total of 170 proteins and 11 069 914 protein-DNA interactions. A web server provides interface for database query. Protein-DNA binding intensities can be retrieved from individual samples for user-provided genomic regions. The retrieved intensities can be used to cluster samples and genomic regions to facilitate exploration of combinatorial patterns, cell-type dependencies, and cross-sample variability of protein-DNA interactions. AVAILABILITY: http://jilab.biostat.jhsph.edu/database/cgi-bin/hmChIP.pl.     

Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq

Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP protocols necessitate the use of large numbers of cells, and library preparation steps associated with current high-throughput sequencing platforms require substantial amounts of DNA; both of these factors preclude the application of ChIP-seq technology to many biologically important but rare cell types. Here we describe a nano-ChIP-seq protocol that combines a high-sensitivity small-scale ChIP assay and a tailored procedure for generating high-throughput sequencing libraries from scarce amounts of ChIP DNA. In terms of the numbers of cells required, the method provides two to three orders of magnitude of improvement over the conventional ChIP-seq method and the entire procedure can be completed within 4 d.     

Active promoters give rise to false positive ‘Phantom Peaks’ in ChIP-seq experiments

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq).We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters.Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.