期刊
NATURE PROTOCOLS
卷 7, 期 6, 页码 1024-1041出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2012.039
关键词
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资金
- Texas STARS
- Alice Kleberg Reynolds Foundation
- Department of the Army [W81XWH04-1-0477]
- Breast Cancer Research Foundation
- Simons Foundation
- American Cancer Society
Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes similar to 3 d from flow cytometry to sequence-ready DNA libraries.
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