Twelve of the 30 markers were successfully genotyped using the partially fragmented DNA extracted from the museum samples (Table S4), so only the corresponding loci from the contemporary samples were included in the data set used for comparison.
Using the MspI fragmented DNA (40 85 μL) described above, the NEBNext End Repair Reaction Buffer (10X; 10 μL), and the NEBNext End Repair Enzyme Mix (5 μL), we incubated the solution (final volume of 100 μl) at 20°C for 30 minutes.
The genomic DNA was sheared using a GeneMachines Hydroshear, and the fragmented DNA processed for cloning into the pSMART vector system (Lucigen, Middleton, WI, USA).
We used AMPure XP to purify the fragmented DNA as follows.
The fragmented DNA was used for library preparation with the NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs) according to manufacturer's instructions.
The ends of the fragmented DNA were repaired using T4 DNA polymerase (New England Biolabs).
Immunoprecipitation of methylated DNA in the fragmented DNA was done using the Methylated DNA IP kit from Zymo Research (catalog number D5101) according to the manufacturer's instructions.
We prepared the fragmented DNA for sequencing using the commercial Illumina protocol for TruSeq DNA library preparations (Illumina, San Diego, CA).
Briefly, the DNA samples (200 μL of 100 500-ng/μL genomic DNA in 0.3 M NaCl) were digested with deoxyribonuclease I (Sigma, St Louis, MO, USA) at 0.0100 500-ng/μL to 105 sec, dependingenomice sample concentration, anDNAhe fragmented DNA was separated using 1% agarose gel electrophoresis.
The fragmented DNA was labelled using 0.86 mM GeneChip DNA labelling reagents (Affymetrix) and 1.5 U/μl terminal deoxy-nucleotidyl transferase (TdT) for 4 hr at 37°C, while the remaining 4.5 μl was examined on 4% TBE agarose gel to confirm that average DNA fragment size was < 180 bp.
