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Multiplexed Illumina sequencing libraries from picogram quantities of DNA

ABSTRACT: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Application of this method allows whole genome studies from samples where material or yields are limiting. Comparison of ChIP-seq libraries constructed from 100 pg DNA (this study) and nanograms of DNA (modENCODE). ChIP antibody: H3K27me3, Active Motif 31955.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Sarah Bowman

PROVIDER: E-GEOD-48431 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Project description:High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Application of this method allows whole genome studies from samples where material or yields are limiting.

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2015-12-15 | E-GEOD-75966 | biostudies-arrayexpress

TELP, a sensitive and versatile library construction method for next-generation sequencing

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Analysis of the Argonaute 4-associated non-coding RNA in Arabidopsis thaliana sheds novel insights into gene regulation through RNA-directed DNA methylation

Project description:RNA-directed DNA methylation (RdDM) is a de novo DNA methylation mechanism in plants that plays a fundamental role in plant defence against invasive DNA and in maintaining genome stability by silencing transposons and repetitive sequences. Using nuclear RNA immunoprecipitation, we constructed a highly enriched library and obtained sequences of ncRNAs specifically associated with Argonaute 4 (AGO4), a key component of RNA-directed DNA methylation. A negative immunoprecipation (IP) library was prepared using IP RNA from tissues that don't express the target protein. A nuclear RNA library was also prepared using nuclei isolated RNA. Three libraries (1X FLAGAGO4 IP, 1X negative IP, 1X nuclear RNA). RNA-IP or nuclear RNA seq cDNA libraries were prepared using the template-switch cDNA library preparation method derived from Zhao et al (2010), and subjected to Illumina GAIIx single end sequencing (100bp)

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2015-02-01 | E-GEOD-55850 | biostudies-arrayexpress

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Simplified ChIP-exo assays II

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