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  • Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species.

Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species.

Plant methods (2012-12-12)
Xin Lin, Leïla Tirichine, Chris Bowler
ABSTRACT

In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Dimethyl Histone H3 (Lys9) Antibody, clone CMA307, clone CMA307, from mouse
Sigma-Aldrich
Anti-dimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
ChIPAb+ Dimethyl-Histone H3 (Lys9) - ChIP Validated Antibody and Primer Set, clone CMA307, from mouse
Sigma-Aldrich
ChIPAb+ Dimethyl-Histone H3 (Lys9) - ChIP Validated Antibody and Primer Set, serum, from rabbit