An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo

Abstract

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell-and tissuespecific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

Distinct regulatory pathways contribute to dynamic CHH methylation patterns in transposable elements throughout Arabidopsis embryogenesis

“…We utilized our recently reported optimized method to profile the genome-wide Arabidopsis embryo methylome at five developmental stages: DAP4, 5, 7, 9, and 12, which correspond to the globular, heart, torpedo, bending torpedo, and mature green stage embryos, respectively ( Figure 1A ) ( Yoo et al., 2021 ). All libraries were made up of at least two biological replicates with robust CT conversion (>99%) and genome coverage (>40X), complying with the recommended standard for methylation data analysis ( Ziller et al., 2015 ) ( Figure 1B ).…”

“…Each released embryo was manually collected using microcapillaries, one by one. All procedures used in this study, including pollination for embryo preparation, sampling of the embryos, genomic DNA extraction, bisulfite library construction, and the processing of whole-genome bisulfite sequencing (WGBS) data, were previously described in detail ( Yoo et al., 2021 ).…”

“…Although DNA methylation changes during plant embryo development have been reported in multiple plant species ( Xing et al., 2015 ; Bouyer et al., 2017 ; Kawakatsu et al., 2017 ; Lin et al., 2017 ; Grover et al., 2020 ; Rajkumar et al., 2020 ), a comprehensive understanding of methylation dynamics for different TE groups or families during embryo development has not been reported. Recently, we described methods for isolating high-purity stage-specific embryos for low-input methylome library construction and data analysis ( Yoo et al., 2021 ). In this study, we used these methods to investigate comprehensive methylome changes in developing Arabidopsis embryos at five stages.…”

Contribution of RdDM to the ecotype-specific differential methylation on conserved as well as highly variable regions between Arabidopsis ecotypes

“…Then, by using Bismark with bowtie2, WGBS reads of Col and Cvi were mapped to the TAIR10 genome without allowing mismatch. All of the other procedures were described in the previous paper [34].…”