New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches
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ABSTRACT:
PROVIDER: PRJNA592170 | ENA |
REPOSITORIES: ENA
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Project description:DNA replication must be faithful and follow a well-defined spatio-temporal program closely linked to genomic organization, transcription, epigenomic marks, intra-nuclear structures, mutation rate and cell fate determination. Replication timing (RT) analyses require complex, precise and time-consuming experimental procedures, and the study of large-size computer files. We improved the RT protocol to speed up and increase its quality and reproducibility. We partly automated the RT protocol via the IP-star robot (Diagenode) and developed a user-friendly software: the START-R suite (Simple Tool for the Analysis of the Replication Timing based on R). START-R suite is an open source software using an R script and an HTML interface to analyse DNA replication timing in a given cell line from microarray or deep-sequencing results. This new approach can be used by every biologist without specific knowledge in bioinformatics and reduces the time required for generating and analyzing data from several samples simultaneously. START-R suite detects constant timing regions (CTR) but also, and it is a novelty, it identifies temporal transition regions (TTR) and significant differences between two experimental conditions. The informatic global analysis requires less than 10 minutes.
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Project description:DNA replication must be faithful and follow a well-defined spatiotemporal program closely linked to transcriptional activity, epigenomic marks, intranuclear structures, mutation rate and cell fate determination. Among the readouts of the spatiotemporal program of DNA replication, replication timing analyses require not only complex and time-consuming experimental procedures, but also skills in bioinformatics. We developed a dedicated Shiny interactive web application, the START-R (Simple Tool for the Analysis of the Replication Timing based on R) suite, which analyzes DNA replication timing in a given organism with high-throughput data. It reduces the time required for generating and analyzing simultaneously data from several samples. It automatically detects different types of timing regions and identifies significant differences between two experimental conditions in ∼15 min. In conclusion, START-R suite allows quick, efficient and easier analyses of DNA replication timing for all organisms. This novel approach can be used by every biologist. It is now simpler to use this method in order to understand, for example, whether 'a favorite gene or protein' has an impact on replication process or, indirectly, on genomic organization (as Hi-C experiments), by comparing the replication timing profiles between wild-type and mutant cell lines.
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Project description:High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an α-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling-based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome. Triplicates of PBC experiments identified a total of 1,975 C-terminal peptides corresponding to 1,190 proteins from 293 T cell line, which is 180% higher than the highest reported number of C-terminal peptides identified from mammalian cells by chemical derivatization-based C-terminomics study. The enrichment efficiency (68%) is the highest among the C-terminomics methods currently reported. In addition, we not only uncovered 50 proteins with truncated C-termini which were significantly enriched in extracellular exosome, vesicle, and ribosome by a bioinformatic analysis but also systematically characterized the whole PTMs on C-terminal in 293 T cells, suggesting PBC as a powerful tool for protein C-terminal degradomics and PTMs investigation. In conclusion, the PBC strategy would benefit high-efficiency and high-throughput profiling of protein C-terminome.
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