GSM1520293: ES-E14 2i pretr.; Mus musculus; MeDIP-Seq
At the end of the trans-differentiation time-course, cells were FACS-purified to remove MEFs, and DNA extracted with phenol-chloroform 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 triplicate. IP triplicates –prepared for each cell type – were pooled and MeDIP DNA libraries were column-purified (MinElute, Qiagen) and eluted in 17μl of EB buffer (pre-warmed at 55ºC). DNA unbound fractions were processed in parallel. MeDIP libraries (and unbound DNA fractions, as control) were amplified by PCR for 12 cycles using distinct combinations of primers: PE 1.0 (Fw) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; PE 2.0 (Rv) CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT; iPCR tag6 (Rv) CAAGCAGAAGACGGCATACGAGATACATTGGCGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC; iPCR tag7 (Rv) CAAGCAGAAGACGGCATACGAGATCAGATCTGGAGA  TCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC; iPCR tag8 (Rv) CAAGCAGAAGACGGCATACGAGATCATCAAGTGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC. Amplified MeDIP libraries (and unbound DNA fractions, as control) 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.
GEO Accession
GSM1520293