Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells
Southall TD, Gold KS, Egger B, Davidson CM, Caygill EE, Marshall OJ and Brand AH (2013) Developmental Cell
Targeted DamID (”TaDa”) is based on DNA adenine methlyltransferase identification (DamID). The Escherichia coli DNA adenine methyltransferase (Dam) protein is fused to a DNA- or chromatin-binding protein of interest and the resulting fusion protein methylates binding sites in the genome. Importantly, DamID requires the Dam-fusion protein to be expressed at extremely low levels to avoid toxicity and nonspecific methylation
In TaDa, the conditional (”targeted”) expression of the Dam-fusion is achieved using the GAL4 system. The introduction of a primary ORF before the Dam-fusion, combined with low rates of translation reinitiation, results in low level expression of the Dam-fusion in a cell-type-specific manner.
TaDa has many advantages: it is robust and reproducible and requires no crosslinking, immunoprecipitation or cell sorting. It can be completed in 3 days from start to finish and can be performed with fewer than 10,000 cells.
Available reagents (require MTA):
Please contact Andrea Brand firstname.lastname@example.org
pUAST-LT3-Dam plasmid plasmid map
pUAST-LT3-Dam-Pol II plasmid plasmid map
UAS-LT3-Dam Drosophila line
UAS-LT3-Dam-Pol II Drosophila line
Reagents for profiling chromatin-binding proteins with TaDa can be found here