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We describe a detailed and widely applicable method for comprehensive proteomic profiling of the fission yeast Schizosaccharomyces pombe by 2-dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry that demonstrates high sensitivity and robust operation. Steps ranging from the preparation of total proteins, digestion of proteins to peptides, and separation...
Regulation of gene expression at the transcriptional level is a fundamental mechanism that is well conserved in all cellular systems. Due to advances in large-scale experimental analyses, we now have a wealth of information on gene regulation such as mRNA expression level across multiple conditions, genome-wide location data of transcription factors and data on transcription factor binding sites....
The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human...
Translation efficiency of an mRNA is related in most cases to its ribosomal association. This association can be readily measured through the separation of cellular complexes on sucrose gradients by velocity sedimentation, and identification of the sedimentation position of the mRNA in the gradient. Since ribosomes are the main driving force for mRNA sedimentation, sedimentation position is highly...
Chromatin immunoprecipitation (ChIP) allows specific protein–DNA interactions to be isolated. Combining ChIP with high-throughput sequencing reveals the DNA sequence involved in these interactions. Here, we describe how to perform ChIP-seq starting with whole tissues or cell lines, and ending with millions of short sequencing tags that can be aligned to the reference genome of the species under investigation...
Analysis of expression quantitative trait loci (eQTL) provides a means for detecting transcriptional regulatory relationships at a genome-wide scale. Here we explain the eQTL analysis pipeline, we introduce publicly available tools for the statistical analysis, and we discuss issues that might complicate the eQTL mapping process. The detection and interpretation of eQTL requires careful consideration...
DNA methylation is a critical epigenetic mark that is essential for mammalian development and aberrant in many diseases including cancer. Over the past decade multiple methods have been developed and applied to characterize its genome-wide distribution. Of these, reduced representation bisulfite sequencing (RRBS) generates nucleotide resolution DNA methylation bisulfite sequencing libraries that enrich...
We describe ribosome affinity purification (RAP), a method that allows rapid purification of ribosomes and associated messages from the yeast Saccharomyces cerevisiae. The method relies on the expression of protein A tagged versions of the ribosomal protein Rpl16, which is used to efficiently recover endogenously formed ribosomes and polysomes from cellular extracts with IgG-coupled spherical microbeads...
The majority of nuclear eukaryotic DNA is packaged into nucleosome cores where DNA is wrapped tightly around histone protein octamers. Such histone bound nucleosomal DNA is less accessible than the short linker DNA between nucleosome cores or the DNA in extended nucleosome free regions. Therefore, the positions of nucleosomes relative to a DNA sequence feature, like a transactivator binding site,...
The ability to quantitatively survey the global behavior of transcriptomes has been a key milestone in the field of systems biology, enabled by the advent of DNA microarrays. While this approach has literally transformed our vision and approach to cellular physiology, microarray technology has always been limited by the requirement to decide, a priori, what regions of the genome to examine. While...
The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator–gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe...
UV cross-linking and immunoprecipitation assay (CLIP) can identify direct interaction sites between RNA-binding proteins and RNAs in vivo, and has been used to study several proteins in tissues and cell cultures. The main challenge of the method is to specifically amplify the low amount of isolated RNA. The current protocol is optimised for efficient RNA purification and ligation of barcoded RNA adapters...
The 3′ poly(A) tail has important roles throughout the eukaryotic mRNA life cycle. A characteristic aspect of poly(A) tail function is furthermore that it can be modulated by changes in its length. This is in turn a well-recognised cellular means to regulate both, mRNA translation and stability, and a positive correlation has often been found between the efficiency of mRNA translation and the length...
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