Different method of isolation of regulatory element from plant
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Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant’s hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes: Oryza sativa, Picea abies, and Crocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by a de novo assembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.
1. Introduction
Chemical information, the majority of the adaptor sequence was removed by PmeI digestion, the rare cutter site (GTTTAAAC) included in the adaptor sequence. 20 μg of the PCR amplifications were digested with PmeI (NEB) in NEB4 buffer and supplemented with 100 ng/μL BSA in two steps. First digestion was performed in a 200 μL reaction volume, containing 200 U PmeI enzyme on 37°C for 2 hours followed by a subsequent volume increase to 250 μL including additional 50 U PmeI and incubated for additional 2 hours. Finally the reaction was stopped at 65°C for 20 minutes.
2.3. Sequencing
The rice and
1. Introduction
Chemical information, the majority of the adaptor sequence was removed by PmeI digestion, the rare cutter site (GTTTAAAC) included in the adaptor sequence. 20 μg of the PCR amplifications were digested with PmeI (NEB) in NEB4 buffer and supplemented with 100 ng/μL BSA in two steps. First digestion was performed in a 200 μL reaction volume, containing 200 U PmeI enzyme on 37°C for 2 hours followed by a subsequent volume increase to 250 μL including additional 50 U PmeI and incubated for additional 2 hours. Finally the reaction was stopped at 65°C for 20 minutes.
2.3. Sequencing
The rice and
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