|For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
MeDIP was performed on 150 ng DNA as described (Borgel et al. 2010) using 1/10 μL anti-5mC antibody for epiblasts and 1/30 μL for PGCs. For epiblasts, we performed one MeDIP with a monoclonal antibody from AbD Serotec (clone 33D3) and one MeDIP with a monoclonal antibody from Abcam (clone 3A3), which showed good reproducibility. For PGCs, we performed two replicates with the AbD Serotec antibody and one replicate with the Abcam antibody. We also performed mock pull-down controls without anti-5mC antibody that did not recover any DNA. Subsequently, 5 ng input DNA and the entire MeDIP product was amplified with the Whole Genome Amplification Kit (Sigma-Aldrich) following the manufacturer's instructions. MeDIP samples were hybridized together with input samples to Roche Nimblegen HD2 2.1M Deluxe Promoter arrays covering −8200 bp to +3000 bp from 23,517 potential TSSs. Sample labeling and microarray hybridization were done according to the standard procedure by Roche Nimblegen. Conversion of DNA with sodium bisulfite was performed with the EpiTect kit (Qiagen). Subsequent analysis by COBRA and sequencing was performed as described (Borgel et al. 2010). Bisulfite PCR reactions in early PGC precursors were performed on the equivalent of at least 50 cells. Clone sequences were processed and aligned with the BISMA software (http://biochem.jacobs-university.de/BDPC/BISMA; Rohde et al. 2010). Primer sequences are given in Supplemental Table 3. Full paper
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