RNA-seq for WT and ddm1(-/-) to evaluate transposon expression in ddm1 1st generation

Silencing of transposons by the chromatin remodeler DDM1 is mediated by the deposition of heterochromatic H2A variants. Transposon mobility and silencing participates in genome evolution but also threaten genome integrity. DECREASED DNA METHYLATION 1 (DDM1) belongs to a conserved family of chromatin remodelers that are required to silence transposons, yet the underlying molecular mechanism has remained unknown. Here we show that DDM1 binds the histone variant H2A.W through two conserved domains that are required to deposit H2A.W and maintain transposon silencing. The mechanism of transcriptional silencing described here is likely shared among chromatin remodelers of the DDM1 family and heterochromatic H2A variants that have evolved in mammals. Total RNA was extracted with Spectrum plant total RNA kit (Sigma Aldrich) from 4-5 week old leaves of WT Col-0 and ddm1 1st generation. For RNA-seq analyses, rRNA was removed by Ribo-Zero rRNA Removal kit (Illumina). After depletion of rRNA, RNA-seq libraries were generated with NEBNext UltraII directional RNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s instructions. The libraries were sequenced with Illumina Hiseq 2500 to generate single-end 50 bp reads. Samples were prepared from three independent biological replicates.