Abstract
Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.
Highlights
Multicellular organisms harbor a single genome, yet exhibit a high degree of cell-type diversity specified by deployment of unique transcriptional networks
PAN-INTACT is a versatile method for isolating cell type specific nuclei from complex, fibrous tissues, such as the adult heart
We found that CM transcripts were highly enriched in nuclei isolated over the 2.2M sucrose cushion and compared favorably with nuclei isolated by Pericentriolar Material 1 (PCM1)-based immunoaffinity purification (Fig 3D)
Summary
Multicellular organisms harbor a single genome, yet exhibit a high degree of cell-type diversity specified by deployment of unique transcriptional networks. Identifying the mechanistic basis for such exquisite cell type specification is a fundamental question in biology and will help illuminate disease pathogenesis. Individual regions within a native organ can be studied using laser capture microdissection (LCM) [5, 6], specific cell types cannot be examined using LCM. FACS is suited for cells in suspension (e.g. cells obtained from whole blood) or soft tissues that are digested (e.g. brain and fetal heart). Many fibrous tissues and most adult organs require harsh digestion conditions to generate a single-cell suspension, which can adversely affect the resulting transcriptional and/or epigenetic profiles
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