Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. We determined DamID scores for Polycomb, normalized by Dam only control, for Drosophila larval central brain, larval fat bodies and repo+ glial cells of larval central brain. All samples were performed with 2 biological replicates. In case of Dam only control for larval central brain, each biological replicate was performed with 3 technical replicates.
Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue.
Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. RNA sequencing of 3 samples, each using 2 biological replicates.
Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue.
Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages.
Project description:Analysis of gene expression regulation typically requires identification of genomic sites where regulatory proteins bind. For this purpose, ChIP and DamID methods applied to cell lines or model organisms are now routinely used, even in selected cell types. In this work, we present modifications to experimental DamID protocol, as well as a custom data processing algorithm that allows to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files thereby making DamID analysis relatively straightforward. Finally, we demonstrate how this pipeline performs when fed with real experimental data.