Sets of ∼35-base ssDNA oligonucleotides (Figs. 1-4 and Supplementary Fig. 1) were purchased (Trilink Biotechnologies, San Diego, CA). Presence of base modifications within these single-stranded oligonucleotides was verified by mass spectrometry. After hybridization and ligation, each end of the resulting dsDNA oligonucleotides was ligated to a hairpin oligonucleotide. Samples were treated with exonucleases to remove any molecules that were not covalently closed. Sequences for the resulting DNA templates, which were 199 bases in length and consisted of a central 84-bp double-stranded region with single-stranded loops at each end, are shown (Supplementary Note).
For sequencing of the full fosmid (Supplementary Figs. 2 and 5), a fosmid clone (clone id: WRM0639cE06) containing an ∼40 kb C. elegans genomic insert was obtained from Geneservice (Cambridge, UK, http://www.geneservice.co.uk/products/clones/Celegans_Fos.jsp) in dam+ E. coli strain EPI300, and cultured and amplified using the inducible origin (CopyControl system, Epicentre, Madison, WI). Fosmid DNA was purified using standard methods. DNA templates were then created directly from fosmid DNA or from whole genome amplified (WGA) fosmid DNA. For WGA libraries, 25 ng of fosmid DNA was amplified using the manufacturer recommended conditions in the GenomiPhi HY DNA Amplification Kit (GE Healthcare, U.K.).
For sequencing of the subsection of the fosmid (Fig. 5 and Supplementary Figs. 3 and 4), an ∼3.7 kb segment (corresponding to positions 12797-16484 within the fosmid) containing 13 instances of the GATC sequence context was PCR amplified from the fosmid using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) and the following primers: Forward 5′-AGTCCTGATGCTTTCACCAAAT-3′; Reverse 5′-ATTTAGATTGCCAAAGCCGTAA-3′. PCR products were cloned into the pCR-Blunt vector using the Zero Blunt PCR Cloning Kit (Invitrogen, Carlsbad, CA) and propagated in the dam+ E. coli strain TOP10 (Invitrogen, Carlsbad, CA). Approximately 25 ng of the DNA was amplified using the REPLI-g Mini Kit (QIAGEN, Valencia, CA) for the generation of the unmethylated control sample.
Fosmid DNA, or an equivalent quantity of WGA fosmid DNA, was sheared to a mean size of 500 bp (Fig. 5 and Supplementary Figs. 3 and 4) or 200 bp (Supplementary Fig. 5) using an ultrasonicator (Covaris Inc, Woburn, MA). Sheared DNA was then end-repaired with a cocktail of T4 DNA polymerase and T4 polynucleotide kinase, purified, and subjected to 3′ A-tailing with Klenow(exo-). The A-tailed fragments were ligated to hairpin oligonucleotides that contained a single 3′ T overhang and 5′ phosphate. Samples were treated with a mixture of exonucleases to remove any molecules that were not covalently closed. The resulting DNA templates were purified using SPRI magnetic beads (AMPure, Agencourt Bioscience, Beverly, MA) and annealed to a two-fold molar excess of a sequencing primer (5′-GGAGGAGGAGGA -3′) that specifically bound to the single-stranded loop region of the hairpin adapters.