title: RRBS of Potassium exposed Jack Pine 12
design: Genomic DNA was cleaved by MspI, targeting sequences with a 5'--C_CGG--3' motif which lead to CpG enrichment. The cleaved fragments were ligated with adenosine tails followed by the addition of sticky ends and sequencing adaptors. Bisulfite conversion was performed to convert unmethylated cytosine to uracil.  DNA from each sample was sequenced and amplified with PCR. Quality control of sequences: Sequence reads were filtered and trimmed with TrimGalore, removing adaptor sequences, sequences with a nitrogen content >5% of the total length, sequences with a Phred score (Error rate) of <20 comprising more than half ot he total length and redundant sequences.  FastQC assessed the quality of clean reads and assigned quality scores to every base. Mapping to reference genome: Sequence reads were aligned to the reference genome of Pinus taeda using Bissmark and hisat 2. Methylation sites were detected based on the base type of the C site alignment from the reference genome using Bismark. Base methylation levels were calculated with the following equation: Methylation level =100*methylated reads/(methylated reads + non-methylated reads).