PAN-INTACT enables direct isolation of lineage-specific nuclei from fibrous tissues

被引:9
作者
Bhattacharyya, Samadrita [1 ]
Sathe, Adwait A. [2 ]
Bhakta, Minoti [1 ]
Xing, Chao [2 ]
Munshi, Nikhil V. [1 ,2 ,3 ,4 ]
机构
[1] UT Southwestern Med Ctr, Dept Internal Med, Div Cardiol, Dallas, TX 75390 USA
[2] UT Southwestern Med Ctr, McDermott Ctr Human Growth & Dev, Dallas, TX 75390 USA
[3] UT Southwestern Med Ctr, Dept Mol Biol, Dallas, TX 75390 USA
[4] Hamon Ctr Regenerat Sci & Med, Dallas, TX 75390 USA
关键词
TRANSLATIONAL PROFILING APPROACH; GENE-EXPRESSION; CELL-TYPES; CHROMATIN; IDENTIFICATION; PURIFICATION; DIVERSITY; ENHANCERS;
D O I
10.1371/journal.pone.0214677
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 cardiomyo-cytes, 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 Nkx2-5(cre/+); R26(sun1-2xsf-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.
引用
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页数:25
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