Whole-genome methylome sequencing (EM-seq) of Arabidopsis thaliana Col-0, nuclear-envelope mutants crwn1–4, crwn1 crwn4 and kaku4 at 12 and 150 days after sowing under short-day conditions
Whole-genome methylome sequencing (EM-seq) of Arabidopsis thaliana Col-0, nuclear-envelope mutants crwn1–4, crwn1 crwn4 and kaku4 at 12 and 150 days after sowing under short-day conditions
Arabidopsis thaliana Col-0 wild type and nuclear-envelope mutants in the Col-0 background were used for EM-seq. The lines included Col-0, crwn1 (SALK_016800), crwn2 (SALK_076653), crwn3 (SALK_099283), crwn4 (SALK_079288), the double mutant crwn1 crwn4, and kaku4 (SALK_076754), together with young reference samples of Col-0 and kaku4-G2 at 12 days after sowing (12 DAS). For the 150 DAS short-day rosette samples, plants were grown on soil under short-day conditions (8 h light / 16 h dark) until 150 days after sowing. From each rosette, two defined leaf cohorts were dissected: • Basal pool: leaves 1–4 (oldest fully expanded rosette leaves) • Apical pool: leaves 11–14 (most recently formed photosynthetic leaves) Basal and apical pools from the same rosette were combined to generate a whole-rosette sample, immediately flash-frozen in liquid nitrogen and stored at −80 °C. Each biological replicate consisted of pooled tissue from 3–4 plants grown in the same pot, and two biological replicates per genotype were collected; a third pot was maintained as backup under identical conditions. For young methylome references, whole 12 DAS seedlings of Col-0 and kaku4-G2 were harvested, pooled per genotype, flash-frozen in liquid nitrogen, and stored at −80 °C. These pooled frozen samples were subsequently split to allow parallel extraction of genomic DNA (for EM-seq) and total RNA (for the RNA-seq experiment) from the same biological material. All Arabidopsis thaliana lines (Col-0 wild type and nuclear-envelope mutants crwn1, crwn2, crwn3, crwn4, crwn1 crwn4, and kaku4) were in the Col-0 background. Seeds were stratified at 4 °C in the dark, then germinated and grown on soil in controlled-environment growth chambers under short-day conditions (8 h light / 16 h dark) for long-term aging experiments, or on plates/soil for 12-day seedling collections. Temperature and humidity were maintained within the standard ranges for Arabidopsis growth, and plants were regularly watered and fertilized according to standard greenhouse practice. For the 150-day sampling, plants were kept continuously under short-day photoperiod to induce strong premature-aging phenotypes in nuclear-envelope mutants prior to EM-seq profiling. Genomic DNA for EM-seq was extracted from the same pooled frozen tissues described in the sample collection protocol (150 DAS rosette basal+apical pools and 12 DAS seedling pools). Tissue was ground to a fine powder in liquid nitrogen using pre-chilled mortars and pestles. High-molecular-weight genomic DNA was isolated using a standard plant genomic DNA protocol, including RNase treatment to remove residual RNA and organic extractions to remove proteins and polysaccharides. DNA was precipitated with alcohol, washed, air-dried, and resuspended in low-EDTA buffer.  DNA yield was quantified fluorometrically (Qubit dsDNA assay) and purity was assessed by spectrophotometry (A260/280 and A260/230 ratios). Integrity was checked by agarose gel electrophoresis. For EM-seq library construction, 200 ng of high-quality genomic DNA per sample was taken from these preparations and used as input for the NEBNext Enzymatic Methyl-seq workflow. For each sample, 200 ng of genomic DNA was used as input for NEBNext Enzymatic Methyl-seq (EM-seq) library construction (NEB E7120). DNA was sheared on a Covaris instrument to an average fragment size of ~250 bp (55 μL volume, peak power 75 W, duty factor 10%, 200 cycles/burst, 160 s). Sheared DNA (50 μL) was subjected to end repair and dA-tailing by adding NEBNext Ultra II End Prep Reaction Buffer and Enzyme Mix (60 μL total) and incubating at 20 °C for 30 min followed by 65 °C for 30 min. EM-seq adaptors were ligated by adding EM-seq Adaptor, Ligation Enhancer, and NEBNext Ligation Master Mix, incubating at 20 °C for 15 min. Adaptor-ligated DNA was purified with SPRI magnetic beads, washed twice with 80% ethanol, and eluted in Elution Buffer to obtain material for enzymatic conversion. After the TET2 oxidation and APOBEC deamination steps (see conversion protocol), converted DNA was purified with SPRI beads and used directly for PCR amplification with EM-seq Index Primers and NEBNext Q5U Master Mix. Libraries were amplified using 10 PCR cycles (98 °C 30 s; 10 cycles of 98 °C 10 s, 60 °C 30 s, 65 °C 60 s; final extension 65 °C 5 min), bead-purified, and eluted in Elution Buffer. Final libraries had fragment size distributions centred around ~300 bp as assessed by Bioanalyzer and were used for Illumina sequencing.