Lambda phage dna

Brassica rapa ssp trilocularis variety R-o-18 were grown in a greenhouse at 70°/60°F (day/night) under at least 16 h of illumination. Plants were fully dried before seed collection. Dry seeds were soaked in water for no more than 60 min before manual dissection to remove mature embryos. Three wild-type or five rdr2 mutant embryos were pooled before DNA extraction with the GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, K0791). Embryos from rdr2/RDR2 heterozygous mothers were individually collected, prepped, and genotyped prior to DNA pooling. rdr2 mutants were used in this study due to their complete loss of 24-nt siRNAs and strong developmental phenotype. Torpedo-stage endosperm and embryo samples were dissected from pistils that were manually pollinated with B. rapa genotype R500. Whole-genome bisulfite sequencing libraries were prepared as previously described [44 (link)]. Lambda Phage DNA (Promega D1521) was included as a bisulfite conversion control. Libraries were pooled and sequenced in a single lane of paired-end 76 nt on an Illumina NextSeq500 at the University of Arizona Genetics Core.

To generate methylated control DNA, 1 μg of lambda phage DNA (Promega) was methylated with CpG methyltransferase SssI (New England BioLabs) for 3 h at 37 °C. Near complete methylation was confirmed by the resistance to methylation-sensitive restriction enzyme HpaII (New England BioLabs). Then, 100 ng of the DNA was bisulfite converted and three lambda loci were amplified by polymerase chain reaction (PCR) (95 °C for 30 s followed by 15 cycles of 95 °C for 30 s, 61 °C for 30 s, and 72 °C for 30 s). The PCR products were cloned into pMD20 (TaKaRa) and sequenced. This analysis demonstrated a 97.9% CpG methylation level (Additional file 1: Figure S4). The PCR primers used are listed in Additional file 1: Table S4.