ONT-seq of Arabidopsis and rice flg22-responsive methylome (5mC and 6mA)
Plant material was ground using mortar and pestle, cooled by liquid nitrogen. For Arabidopsis, three biological replicates per time point (12 and 72 hours post Flg22) and treatment (mock and Flg22) were processed for sequencing, while for rice only one biological replicate was used per time point (12 and 72 hours post Flg22 versus mock). High molecular weight (HMW) DNA was extracted using the NucleoBond HMW DNA kit (MN740160.20, Macherey Nagel) according to the manufacturers instructions. DNA quality and size distribution were assessed using the HS Genomic DNA 50 kb kit on the Fragment Analyzer 5200 (DNF-468-0500, Agilent Technologies). Concentration of the genomic DNA was measured with the Qubit dsDNA HS Assay Kit (Q32851, Thermo Fisher Scientific). For Arabidopsis and rice samples, respectively, 400 ng and 600 ng extracted HMW genomic DNA was used as input for the library preparation with the Native Barcoding Kit v14 (SQK_NBD114.24, Oxford Nanopore Technologies), following manufacturers instructions with some minor adaptations. Specifically, after the Native Barcoding Ligation step, samples were each purified separately with Ampure XP beads (ratio 0.4x; A63881, Beckman Coulter) and eluted in 8 l molecular biology grade water before pooling them for the Native Adapter ligation step. In addition, pooling was performed using an equimolar mass of each purified sample, calculated from Qubit concentration measurements and Fragment Analyzer size profiles obtained after clean-up. All nine Arabidopsis samples were pooled together into a single library. Rice samples were pooled into two libraries, each comprising one mock- and one Flg22-treated sample collected at the same time point (12 or 72 hpt). The prepared library pools were entirely, with a maximum of 30 fmol, loaded into a PromethION flow cell (FC) (R10.4.1) on a PromethION sequencer (ONT). After 1-2 days, the FC was washed using the Flow Cell Wash Kit (EXP-WSH004, ONT) to recover pore activity, and a fresh library was loaded. The FC was then run to exhaustion.