Project description:Chromatin structure and transcription factor localization can be assayed genome-wide by sequencing genomic DNA fractionated by protein occupancy or other properties. However, current technologies involve multiple steps that introduce bias and inefficiency. Here we apply a single-molecule approach to directly sequence chromatin immunoprecipitated DNA with minimal sample manipulation. This method is accurate, compatible with just 50 picograms of DNA and should thus facilitate charting chromatin maps from limited cell populations. Application of a single-molecule approach to directly sequence chromatin immunoprecipitated DNA of the CTCF DNA binding protein.
Project description:The diversity and complexity of the microbiome's genomic landscape are not always mirrored in its proteomic profile. Despite the anticipated proteomic diversity, observed complexities of microbiome sample are often lower than expected. Two main factors contribute to this discrepancy: limitations in mass spectrometry's detection sensitivity and bioinformatics challenges in metaproteomics identification. This study introduces a novel approach to evaluating sample complexity directly at the full mass spectrum (MS1) level rather than relying on peptide identifications. When analyzing under identical mass spectrometry conditions, microbiome samples displayed significantly higher complexity, as evidenced by the spectral entropy and peptide candidate entropy, compared to single-species samples. The research provides solid evidence for the complexity of microbiome in proteomics indicating the optimization potential of the bioinformatics workflow.
Project description:The present study was conducted in the frame of the EU-funded Graphene Flagship project. We previously evaluated the impact of graphene oxide (GO) on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild-type versus AhR-deficient zebrafish. Here, we performed single-cell RNA-sequencing (10x Genomics) on whole (dissociated) germ-free (GF) zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.