Methods in molecular biology
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DNA methylation changes are dynamic processes which occur at cytosines of CpG dinucleotides and contribute to normal development but also to diseases. DNA methylation changes are most effective in promoters and enhancers, the former frequently being CpG-rich and the latter, in contrast, CpG-poor. Many genome-wide methods for DNA methylation analysis interrogate predominantly CpG-rich regions and, hence, spare enhancers and other potentially important genomic regions. ⋯ In a subsequent step, the non-covalently bound adapter oligonucleotide needs to be replaced by a novel oligonucleotide to provide the proper adapter sequence for the reverse strand in paired-end sequencing. The presented protocol describes an improved, simplified version of TWGBS where the inefficient oligo-replacement is circumvented by usage of a sequencing-compatible transposase-adapter complex. Consequently, genomic DNA of only a few hundred human cells is required to interrogate the complete human DNA methylome.
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The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.
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The emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology provides tools for researchers to modify genomes in a specific and efficient manner. The Type II CRISPR-Cas9 system enables gene editing by directed DNA cleavage followed by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Here, we described the use of the Type II CRISPR-Cas9 system in detail from designing the guides to analyzing the desired gene disruption events.
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ProStaR is a software tool dedicated to differential analysis in label-free quantitative proteomics. Practically, once biological samples have been analyzed by bottom-up mass spectrometry-based proteomics, the raw mass spectrometer outputs are processed by bioinformatics tools, so as to identify peptides and quantify them, by means of precursor ion chromatogram integration. ⋯ To achieve this statistical step, it is possible to rely on ProStaR, which allows the user to (1) load correctly formatted data, (2) clean them by means of various filters, (3) normalize the sample batches, (4) impute the missing values, (5) perform null hypothesis significance testing, (6) check the well-calibration of the resulting p-values, (7) select a subset of differentially abundant proteins according to some false discovery rate, and (8) contextualize these selected proteins into the Gene Ontology. This chapter provides a detailed protocol on how to perform these eight processing steps with ProStaR.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.