Genomic DNA was extracted according to Smith et al. (2009) (link), except for sperm DNA (Jeffreys et al. 1994 (link)). The Epitect kit (Qiagen) was used for bisulfite conversion of DNA, except for limited quantities (oocytes and embryos from E3.5 to E6.5) where DNA was embedded in agarose beads before processing for bisulfite conversion according to Proudhon et al. (2012) (link). Nested or seminested PCR was performed using the primers listed in Supplemental Table S1. PCR amplicons were gel-purified (Qiagen) and subsequently cloned in pCR2.1 Topo-TA vector (Invitrogen) before Sanger sequencing of at least 30 clones. BiQ Analyzer software was used for sequence alignments (Bock et al. 2005 (link)).
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Bisulfite Conversion and Sequencing of Genomic DNA
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Corresponding Organization :
Other organizations : Institut Curie, Centre National de la Recherche Scientifique, Inserm, Université de Bourgogne
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Variable analysis
independent variables
- DNA extraction protocol (Smith et al. 2009 vs. Jeffreys et al. 1994)
- Bisulfite conversion protocol (Epitect kit vs. Proudhon et al. 2012)
- DNA methylation patterns (determined by Sanger sequencing of cloned PCR amplicons)
- Nested or seminested PCR using the primers listed in Supplemental Table S1
- DNA sequence alignment using BiQ Analyzer software (Bock et al. 2005)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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