title: GSM6156890: MBD_input_LU_jj8; Canis lupus familiaris; MBD-Seq
library_construction_protocol: Beagle tissues (11 types, ~25mg each) were incubated in Buffer ATL (Qiagen, USA) and protease K at 56°C until the tissue was completely lysed. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, USA), according to the manufacturer's protocol with modifications. The concentration and purity of isolated genomic DNA was assessed by NanoDrop. For each sample, 3 μg of DNA, normalized to 20 ng/μl, was subjected to shearing with the Bioruptor® Pico (Diagenode) and the size of fragmented DNA (~300 bp) was verified on an agarose gel. Finally, the concentration of sheared double-stranded DNA was measured using Qubit dsDNA HS Assay (ThermoFisher Scientific, USA).  For MBD-enrichment, 500 ng of fragmented double-stranded DNA was enrichedwith methylated CpGs using MethylMiner™ Kit (Invitrogen, cat# ME10025), according to the manufacturer's instructions. To counteract the possibility of increased unspecific binding, we used more stringent wash conditions. In more detail, for each capture reaction, prepared MBD-beads (10 μg beads and 350 ng MBD-biotin protein were used in the same reaction) were added to each 500 ng of fragmented DNA input. Prepared MBD-beads were diluted to 1x in Bind/Wash Buffer prior to addition of each DNA sample, to increase pipetting accuracy. Each capture reaction was brought up to 200 μl final volume in 1x Bind/Wash Buffer, and incubated on a rotator for 40 min at RT. Following incubation, each tube was placed on a magnet for 1 minute and the supernatant containing the non-captured (unmethylated) DNA fragments was removed. The beads with bound methylated DNA were then washed twice by incubation with 200 μl Bind/Wash Buffer. The beads were washed with Bind/Wash buffer, according to the protocol, and eluted in step-wise elution using 200 μl of serially diluted elution buffer (200, 300, 400, 600, and 800 mM). Eluted methylated DNA fragments in the 600 and 800 mM elution buffers underwent cluster generation and 200 bp paired-end sequencing using TruSeq Nano DNA Kit on the Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA).