Marija Kundakovic1, Yan Jiang1, David H Kavanagh2, Aslihan Dincer2, Leanne Brown1, Venu Pothula1, Elizabeth Zharovsky1, Royce Park1, Rivka Jacobov1, Isabelle Magro1, Bibi Kassim1, Jennifer Wiseman1, Kristen Dang3, Solveig K Sieberts3, Panos Roussos2, Menachem Fromer4, Brent Harris5, Barbara K Lipska6, Mette A Peters3, Pamela Sklar2, Schahram Akbarian7. 1. Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York. 2. Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York; Friedman Brain Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York. 3. Sage Bionetworks, Seattle, Washington. 4. Friedman Brain Institute, and Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York. 5. Department of Neurology, Georgetown University Medical Center, Washington, DC; Human Brain Collection Core, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland. 6. Human Brain Collection Core, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland. 7. Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address: Schahram.akbarian@mssm.edu.
Abstract
BACKGROUND: The nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed. METHODS: Protocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries. RESULTS: We present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms. CONCLUSIONS: Our practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.
BACKGROUND: The nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed. METHODS: Protocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries. RESULTS: We present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms. CONCLUSIONS: Our practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.
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