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Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP).
Gryder, Berkley E; Khan, Javed; Stanton, Benjamin Z.
Afiliação
  • Gryder BE; Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. berkley.gryder@nih.gov.
  • Khan J; Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. khanjav@nih.gov.
  • Stanton BZ; Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA. benjamin.stanton@nationwidechildrens.org.
Nat Protoc ; 15(3): 1209-1236, 2020 03.
Article em En | MEDLINE | ID: mdl-32051612
Methods developed to capture protein-anchored chromatin interactions (chromatin interaction analysis by paired-end tag sequencing and HiChIP) have yielded tremendous insights into the 3D folding principles of the genome, but are normalized by sequencing depth and therefore unable to accurately measure global changes in chromatin interactions and contact domain organization. We herein describe the protocol for absolute quantification of chromatin architecture (AQuA)-HiChIP, an advance that allows the absolute differences in protein-anchored chromatin interactions between samples to be determined. With our method, defined ratios of mouse and human fixed nuclei are mixed and subjected to endonuclease digestion. Chromatin contacts are captured by biotin-dATP incorporation and proximity ligation, followed by gentle shearing, ChIP, biotin capture and paired-end sequencing. 3D contacts are counted from paired-end tags (PETs) from the human genome and are normalized to the total PETs from the mouse genome. As orthogonal normalization allows observation of global changes, the approach will enable more quantitative insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory. With our approach, we have discovered that rapid histone deacetylase inhibition disrupts super enhancer function by creating many new aberrant contacts. The code for data analysis is available at https://github.com/GryderArt/AQuA-HiChIP. This protocol reports both experimental and bioinformatic details to perform AQuA-HiChIP, going from cell culture to ranking chromatin interactions within 6 d.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Cromatina / Análise de Sequência de DNA / Imunoprecipitação da Cromatina Limite: Animals / Humans Idioma: En Revista: Nat Protoc Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Cromatina / Análise de Sequência de DNA / Imunoprecipitação da Cromatina Limite: Animals / Humans Idioma: En Revista: Nat Protoc Ano de publicação: 2020 Tipo de documento: Article