Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: identifying ChIP-quality p300 monoclonal antibodies. 2014

William C Gasper, and Georgi K Marinov, and Florencia Pauli-Behn, and Max T Scott, and Kimberly Newberry, and Gilberto DeSalvo, and Susan Ou, and Richard M Myers, and Jost Vielmetter, and Barbara J Wold
1] Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA [2].

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1.

UI MeSH Term Description Entries
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D006657 Histones Small chromosomal proteins (approx 12-20 kD) possessing an open, unfolded structure and attached to the DNA in cell nuclei by ionic linkages. Classification into the various types (designated histone I, histone II, etc.) is based on the relative amounts of arginine and lysine in each. Histone,Histone H1,Histone H1(s),Histone H2a,Histone H2b,Histone H3,Histone H3.3,Histone H4,Histone H5,Histone H7
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D001331 Automation Controlled operation of an apparatus, process, or system by mechanical or electronic devices that take the place of human organs of observation, effort, and decision. (From Webster's Collegiate Dictionary, 1993) Automations
D012371 Robotics The application of electronic, computerized control systems to mechanical devices designed to perform human functions. Formerly restricted to industry, but nowadays applied to artificial organs controlled by bionic (bioelectronic) devices, like automated insulin pumps and other prostheses. Companion Robots,Humanoid Robots,Remote Operations (Robotics),Social Robots,Socially Assistive Robots,Telerobotics,Soft Robotics,Assistive Robot, Socially,Companion Robot,Humanoid Robot,Operation, Remote (Robotics),Operations, Remote (Robotics),Remote Operation (Robotics),Robot, Companion,Robot, Humanoid,Robot, Social,Robot, Socially Assistive,Robotic, Soft,Social Robot,Socially Assistive Robot,Soft Robotic
D014157 Transcription Factors Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process. Transcription Factor,Factor, Transcription,Factors, Transcription
D017422 Sequence Analysis, DNA A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis. DNA Sequence Analysis,Sequence Determination, DNA,Analysis, DNA Sequence,DNA Sequence Determination,DNA Sequence Determinations,DNA Sequencing,Determination, DNA Sequence,Determinations, DNA Sequence,Sequence Determinations, DNA,Analyses, DNA Sequence,DNA Sequence Analyses,Sequence Analyses, DNA,Sequencing, DNA
D047369 Chromatin Immunoprecipitation A technique for identifying specific DNA sequences that are bound, in vivo, to proteins of interest. It involves formaldehyde fixation of CHROMATIN to crosslink the DNA-BINDING PROTEINS to the DNA. After shearing the DNA into small fragments, specific DNA-protein complexes are isolated by immunoprecipitation with protein-specific ANTIBODIES. Then, the DNA isolated from the complex can be identified by PCR amplification and sequencing. Immunoprecipitation, Chromatin
D050881 E1A-Associated p300 Protein A member of the p300-CBP transcription factors that was originally identified as a binding partner for ADENOVIRUS E1A PROTEINS. p300 E1A-Associated Coactivator,E1A Associated p300 Protein,E1A-Associated Coactivator, p300,p300 E1A Associated Coactivator,p300 Protein, E1A-Associated

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