Repli-Seq
Nascent DNA Replication Strand Sequencing
Repli-Seq maps the sequences of nascent DNA replication strands throughout the whole genome during each of the 6 cell-cycle phases (Hansen et al., 2010). This process is achieved by growing cells in media containing BrdU to replace thymidine. The cells are sorted to their current state in cell division using fluorescence-activated cell sorting (FACS). BrdU-labeled DNA strands are immunoprecipitated by anti-BrdU antibodies on magnetic beads. These immunoprecipitated strands can be prepared for sequencing following the TruSeq DNA library preparation protocol.
Advantages:
- Maps sequences of newly replicated DNA to the phases of cell division
- Low sample input required (5000 cells) makes it suitable for studying rare cell populations
- Streamlined DNA library preparation step
Disadvantages:
- Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Reuter J. A., Spacek D. V. and Snyder M. P. High-throughput sequencing technologies. Mol Cell. 2015;58:586-597
References:
Ohbayashi R., Watanabe S., Ehira S., Kanesaki Y., Chibazakura T. and Yoshikawa H. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution. ISME J. 2015;
Deyle D. R., Hansen R. S., Cornea A. M., et al. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol. 2014;21:969-975
Barlow J. H., Faryabi R. B., Callen E., et al. Identification of early replicating fragile sites that contribute to genome instability. Cell. 2013;152:620-632
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History: Repli-Seq
Revision by sbrumpton on 2017-06-21 07:50:21 - Show/Hide
Nascent DNA Replication Strand Sequencing
Repli-Seq maps the sequences of nascent DNA replication strands throughout the whole genome during each of the 6 cell-cycle phases (Hansen et al., 2010). This process is achieved by growing cells in media containing BrdU to replace thymidine. The cells are sorted to their current state in cell division using fluorescence-activated cell sorting (FACS). BrdU-labeled DNA strands are immunoprecipitated by anti-BrdU antibodies on magnetic beads. These immunoprecipitated strands can be prepared for sequencing following the TruSeq DNA library preparation protocol.
Advantages:- Maps sequences of newly replicated DNA to the phases of cell division
- Low sample input required (5000 cells) makes it suitable for studying rare cell populations
- Streamlined DNA library preparation step
Disadvantages:- Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides
Reagents:Illumina Library prep and Array Kit SelectorReviews:Reuter J. A., Spacek D. V. and Snyder M. P. High-throughput sequencing technologies. Mol Cell. 2015;58:586-597References:Ohbayashi R., Watanabe S., Ehira S., Kanesaki Y., Chibazakura T. and Yoshikawa H. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution. ISME J. 2015; Deyle D. R., Hansen R. S., Cornea A. M., et al. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol. 2014;21:969-975Barlow J. H., Faryabi R. B., Callen E., et al. Identification of early replicating fragile sites that contribute to genome instability. Cell. 2013;152:620-632