Designing Cell-Type-Specific Genome-wide Experiments Ava Handley, Tamás Schauer, Andreas G. Ladurner, Carla E. Margulies  Molecular Cell  Volume 58, Issue 4, Pages 621-631 (May 2015) DOI: 10.1016/j.molcel.2015.04.024 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Overview of Workflow Used to Obtain Cell-Type-Specific Genome-wide Data All cell-type-specific genomics analyses follow a similar workflow. The cell type of interest needs to be identified and labeled (step 1). The chromatin or RNA from that cell type is then isolated and assayed (steps 2 and 3). Computational analysis of the data reveals cell-type-specific features that are experimentally validated (steps 4 and 5). Molecular Cell 2015 58, 621-631DOI: (10.1016/j.molcel.2015.04.024) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Methods for the Cell-Type-Specific Purification of Whole Cells Manual and FACS methods depend on protease dissociation of cells. For manual isolation, GFP-positive cells are separated manually from non-fluorescent cells under a fluorescence-dissecting microscope. The FACS machine separates cells based on fluorescence. For LCM, a thin, transparent film becomes adhesive when irradiated by an infrared laser that is placed over the cryosectioned tissue. After identifying the cell of interest by staining, immunohistochemistry, or GFP signal, an infrared laser focused over the cell of interest causes the cell(s) to adhere to the film, allowing the cell(s) of interest to be selectively removed from the tissue section. Molecular Cell 2015 58, 621-631DOI: (10.1016/j.molcel.2015.04.024) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Methods for Isolating Cell-Type-Specific Nuclei (A) Affinity tags commonly used for isolating cell-type-specific nuclei. (B) For both FACS-based sorting of nuclei and the INTACT method, nuclei are prepared from the tissue of interest. GFP-based fluorescence and immunohistochemistry of either endogenous or transgenically tagged nuclear localized markers have been used for isolating nuclei via a FACS machine. For INTACT, tagged proteins localized to the outer nuclear envelope are used to biochemically purify nuclei using magnetic beads. Molecular Cell 2015 58, 621-631DOI: (10.1016/j.molcel.2015.04.024) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Biochemical Isolation of Cell-Type-Specific RNA and Chromatin Tagged RNA binding proteins are expressed in a specific cell type. After the tissue is homogenized, the tagged RNA binding proteins and the associated RNAs are immunopurified. A related approach is used for chromatin using CAST-ChIP, except that the tagged proteins bind chromatin, and associated DNA is biochemically isolated. TU tagging allows cell-type-specific RNAs to be directly isolated. By expressing the protozoan enzyme UPRT in specific cells, 4TU is incorporated into RNA. After homogenizing the tissue and RNAs are isolated, the 4TU-labeled RNA is biotinylated and streptavidin purified. In TaDa, expressing a bacterial sequence-specific DNA methylase fused to metazoan RNA polymerase II subunit chemically tags the DNA that RNA polymerase II binds to with a methylation marker. The chromatin is then isolated and digested with the methylation-specific restriction enzyme Dpn1. The methylated DNA is then amplified and sequenced, providing an indirect measurement of where RNA polymerase II was associated with. Molecular Cell 2015 58, 621-631DOI: (10.1016/j.molcel.2015.04.024) Copyright © 2015 Elsevier Inc. Terms and Conditions