GSM7770127: WT met1-3 F3 2a; Arabidopsis thaliana; OTHER
DNA was extracted using a CTAB method Leaf tissue from 28-day old plants was collected and flash frozen in liquid nitrogen. Two leaves per sample were ground using a Qiagen TissueLyser II and immediately after 200 µl of CTAB was added and heated to 65°C for approximately 20 minutes. This was followed by the addition of 200 µl chloroform, 10 minute centrifugation and ethanol washes to isolate total genomic DNA. The DNA concentration was quantified using a Qubit Flex fluorometer, and a diluted spike-in was added according to NEB recommendations (1 µl diluted pUC19 (0.001 ng) control DNA and 1 µl diluted unmethylated lambda DNA (0.02 ng) in 0.1X TE, pH 8.0 per 10–200 ng plant sample DNA). Genomic DNA samples with added spike-ins were sonicated to an average fragment size of 300bp using a Covaris S220 Focused Ultrasonicator (80s treatment, 140 peak power, 10 duty factor 200 cycles/burst). Subsequently, samples were incubated with an RNAse cocktail and RNAse was removed using a Qiagen spin column. EM-seq libraries were constructed following the NEBNext® Enzymatic Methyl-seq Kit protocol. Library quality was verified with a KAPA Library Quant Kit and fragment analyzer. The samples were pooled to a concentration of 4 ng/µl and 19.2 nM and sequenced using an Illumina NextSeq 2000 (paired-end 150 x 150 bp) 11 million average reads/sample.