Methylome Profiling Using Illumina Sequencing

The genome was first digested with the PstI restriction enzyme (Thermo Scientific; Waltham, MA, USA) in a suitable range (≈450 bp) for Illumina (San Diego, CA, USA) sequencing [26 (link)]. Then, illumina sequencing barcodes are ligated to each end of the digested DNA fragments, allowing the pool of DNA samples to be immunoprecipitated together. Each pooled DNA sample contains different barcodes identifying each individual reduced genome. Then, a 50 ng fraction of the DNA pool, representing the genetic background of the libraries, hereby called inputs, was amplified by PCR. Then, the methylated fraction of the sampled DNA was captured by an anti-methyl-cytosine antibody (Diagenode, Sparta, TN, USA) [29 (link)]. After this step, the methylated DNA was amplified using PCR, which is followed by a clean-up of the primer dimers and unbound adapters [79 (link),80 (link)]. The procedure generated a library that corresponds to the methylated portion of the reduced genome. The libraries were quantified, clustered, and end-paired sequenced in the Illumina NovaSeq6000 platform with a read length of 150bp at the SNP & SEQ facilities of the SciLifeLab (Uppsala, Sweden).