Anti methyl cytosine antibody

To analyse DNA methylation in the reduced genomic fractions of many individuals, we combined genotyping by sequencing (GBS) [83 (link)] with methylated DNA immunoprecipitation (MeDIP) [84 (link)], as previously described [85 (link)]. Briefly, the individual DNA samples are first digested with the PstI (ThermoFisher Scientific) restriction enzyme, whose recognition site does not contain CpGs. Then, Illumina adapters and individual barcodes were added to the fragments produced after this digestion of individual DNA samples [86 ] to later identify individual fragment samples bioinformatically [86 ,87 (link)]. The individually barcoded samples are then pooled and subsequently subjected to enrichment of the methylated fraction (MeDIP) by an anti-methyl-cytosine antibody (2 μg/μl; catalogue number C15200006; Diagenode, Denville, NJ, USA), as previously described [84 (link)]. PCR was then performed after the MeDIP capture of the methylated fraction of the pool of barcoded DNA samples to create the sequencing library [85 (link)]. Paired-end sequencing was performed with a read length of 100 bp on the Illumina HiSeq2500 platform, at the Animal Biotechnology Laboratory (ESALQ/USP), Brazil.

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).