A sodium bisulphite DNA modification protocol was used as described previously (7 (link)). The example data were derived from bisulphite sequencing of the differentially methylated region (DMR) in the imprinted ZAC (PLAGL1) tumour-suppressor gene (7 (link)). The reference sequence corresponds to nucleotides 48434376–48433838 of the Chromosome 6 assembly, accession NT_025741.14. PCR of bisulphite-modified DNA was carried out using the following primers, which amplify both methylated and unmethylated sequences: Zac9: dCCCAACCRTATCTAAATCAAAACT; and Zac1: dGTGTTTAGGATAGTGTTTGGTT. PCR conditions were as follows: denaturation at 94°C for 3 min followed by 35 cycles of 94°C, 30 s; 58°C, 30 s; 72°C, 30 s and a final extension step at 72°C for 3 min. PCR products were gel-extracted, purified using the Geneclean II kit (MP Biomedicals, Solon, OH, USA) and ligated into pGEM-T Easy vector (Promega, Madison, WI, USA) according to the manufacturer's protocol. Ligations were transformed into DH5α cells and DNA extracted from recombinant clones using the Qiaprep Spin miniprep kit (Qiagen, Valencia, CA, USA).