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Location Analysis and Expression Profiling Using Next-Generation Sequencing for Research in Neurodegenerative Diseases

Gotovac K, Borovečki F, Habek M

Neurodegenerative diseases carry a huge burden in terms of human suffering and economic cost. In spite of advances in the field of neurology, there is still no effective cure. A combination of novel genomic approaches and techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq that enable high-quality data could provide better understanding of transcriptional and epigenetic signatures useful for both biomarker development and drug discovery in neurodegenerative diseases. This chapter provides detailed protocols of the different steps required to generate a successful ChIP-seq and RNA-seq library. ChIP-seq protocol starts with cell collection and fixation, chromatin preparation, immunoprecipitation, and finally library preparation. RNA-seq protocol described in this chapter starts with the isolation of peripheral blood mononuclear cells (PBMCs), RNA isolation from cells, and library preparation. The ChIP-seq protocol is optimized for human neuroglioma cells (H4), and RNA-seq protocol is optimized for PBMCs, but both protocols can be adapted to different cell types with minor modifications. The obtained libraries are suitable for sequencing on Illumina GAIIx platform.

Chromatin Shearing

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