GSM1568789: 4F iTSC clone B; Mus musculus; MeDIP-Seq
DNA extracted according to standard protocols. DNA was used for MeDIP analysis as described previously (Senner et al., Stem Cells 2012). Genomic DNA was fragmented by sonication (Diagenode Bioruptor), and adaptor ligated with paired-end adaptors 5’-PGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3’ and 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ (* Phosphorothioate group) and the NEBnext library preparation kit. Subsequently immunoprecipitaions with an anti-5-methylcytosine antibody were carried out in duplicate. IP duplicates –prepared for each sample – were pooled and MeDIP DNA libraries were column-purified (MinElute, Qiagen). MeDIP libraries were amplified by PCR for 12 cycles using distinct combinations of primers: PE 1.0 (Fw) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT, and various iPCR tag (Rv) primers (Quail et al, 2012) Amplified MeDIP libraries were  resolved by 2% agarose gel electrophoresis, and DNA fragments within the 300-500 nt range were cut out and DNA was purified using the Qiagen Gel extraction kit. 5mC immuno-precipitation efficiency was confirmed by analysing ES and TS  cell MeDIP pre- and post-amplification libraries by quantitative PCR for candidate loci (Elf5, Nanog). Library concentration was determined using KAPA Illumina SYBR Universal Lid Q Kit (ANACHEM) and by Bioanalyzer.