GSM1233310: h_BrainA2_MBD_M; Homo sapiens; OTHER
DNA was extracted and sheared to approximately 400 bp using sonication and then ligated to Illumina's adapters. Methylated DNA was purified by two step MBD Affinity Purification as described in Skene et al., 2010. Libraries were prepared as follows using enzymes from New England Biolabs (NEB). End Repair was carried out by incubating fragmented DNA with T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase for 30 minutes at 20 C. The blunt, phosphorylated ends were treated with Klenow fragment (exo-) and dATP for 30 minutes at 37 C to yield a protruding 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. Ligated DNA was ethanol precipitated and then reconstituted in running buffer prior to MAP-purification. Purified DNA was PCR amplified with Illumina primers for 10-12 cycles. Library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated by gel extraction. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a Genome Analyzer following the manufacturer's protocols. Sequencing was performed at the Wellcome Trust Sanger Institute; Hinxton, Cambridge, UK.
GEO Accession
GSM1233310