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.

Global RNA half-life analysis in Escherichia coli reveals positional patterns of transcript degradation.

Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-type Escherichia coli MG1655. RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons. The half-life of total mRNA was 6.8 min under the conditions tested. We also observed significant relationships between gene functional assignments and transcript stability. Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition. This supports a novel mechanism of transcription for the tdc operon, whose promoter lacks any recognizable sigma binding sites. Probe by probe analysis of all known and predicted operons showed that the 5' ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3' direction, supporting and further generalizing the current model of a net 5' to 3' directionality of degradation. Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive. We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants. The 16 ORF dcw gene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation. We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.

Transcriptome analysis of Escherichia coli using high-density oligonucleotide probe arrays.

Microarrays traditionally have been used to analyze the expression behavior of large numbers of coding transcripts. Here we present a comprehensive approach for high-throughput transcript discovery in Escherichia coli focused mainly on intergenic regions which, together with analysis of coding transcripts, provides us with a more complete insight into the organism's transcriptome. Using a whole genome array, we detected expression for 4052 coding transcripts and identified 1102 additional transcripts in the intergenic regions of the E.coli genome. Further classification reveals 317 novel transcripts with unknown function. Our results show that, despite sophisticated approaches to genome annotation, many cellular transcripts remain unidentified. Through the experimental identification of all RNAs expressed under a specific condition, we gain a more thorough understanding of all cellular processes.