The detailed ChIP protocol is in the Supplemental Material . For sequencing, DNA fragments were made into libraries using the NEBNext Ultra II FS kit (New England Biolabs). Libraries were pooled and sequenced on a NextSeq 500 platform (Illumina) with paired-end 150-nt runs at high output (developing seeds) or single-end 75-nt runs at high output (mature embryos). Adaptor sequences were removed using Trimmomatic (Bolger et al. 2014 (link)); trimmed reads were then aligned on the Arabidopsis TAIR10 genome using Bowtie2 (Langmead and Salzberg 2012 (link)). Duplicated reads are removed using the Picard Tool suite, and coverage was calculated and mapped to DMRs using Bedtools. All distributions were compared with the first WT sample distribution using the Kolmogorov–Smirnov test with R.
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ChIP-Seq Analysis of Arabidopsis
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Corresponding Organization :
Other organizations : Agriculture and Agri-Food Canada, Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Université de Montpellier, Institut de Recherche pour le Développement
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Variable analysis
independent variables
- Sequencing platform (NextSeq 500 vs Illumina)
- Sequencing read length (paired-end 150-nt vs single-end 75-nt)
- Mapped sequencing reads
- Coverage of differentially methylated regions (DMRs)
- Arabidopsis TAIR10 genome used for read alignment
- Trimmomatic used for adapter removal
- Bowtie2 used for read alignment
- Picard Tool suite used for duplicate read removal
- WT sample distribution used as a comparison for all other distributions
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