Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

Abstract

SummaryThe ability to profile transcription and chromatin binding in a cell type-specific manner is a powerful approach for understanding cell fate specification and cellular function in multicellular organisms.We recently developed Targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA-and chromatin-binding proteins in vivo without cell isolation. As a Protocol Extension, this article describes substantial modifications to an existing Protocol and offers additional applications.TaDa builds upon DamID, a technique for detecting genome-wide DNA binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution.TaDa ensures that Dam-fusion proteins are expressed at very low levels, avoiding toxicity and potential artefacts from over-expression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to Nextgeneration Sequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared to other methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II, TaDa can also identify transcribed genes in a cell type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure -from collecting tissue samples to generating sequencing libraries -can be accomplished within 5 days.

Mechanosensory neuron regeneration in adult Drosophila

“…In our case, the low-levels of JO self-division and the sensitivity to resolve singleneurons by time-lapse imaging on individual flies provides an in vivo scalable platform to screen for smallmolecules that enhance JO regeneration (Fernández-Hernández et al, 2016). For selected targets, the transcriptomic and epigenetic changes on JO neurons can be assessed as they occur in vivo, by geneticallyencoded available tools (Marshall et al, 2016;Marshall and Brand, 2017;Southall et al, 2013). Further, functional contribution of regenerated JO can be readily assessed by established behavioral protocols (Kamikouchi et al, 2009;Sun et al, 2009;Vaughan et al, 2014).…”

Persistent Epigenetic Reprogramming of Sweet Taste by Diet

“…For according to protocol (Marshall et al, 2016) , and subsequently purified. Purified DNA was digested with DpnII followed by sonication to yield fragments averaging 200-300bp.…”

“…Purified DNA was digested with DpnII followed by sonication to yield fragments averaging 200-300bp. TaDa adaptors were removed from sonicated DNA by digestion (Marshall et al, 2016) .…”

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

“…Another strategy is to consider techniques that may be more sensitive and detect lower signals (or infrequent binding), as might be observed with “hit and run” repressors. Techniques that involve enzymatic mechanisms, such as DNA adenine methyltransferase identification (DamID)‐seq, may be particularly sensitive. DamID also has other advantages such as not relying on antibodies and thus avoiding epitope masking.…”