Agilent bioanalyzer analysis

All sample preparation and MeDIP-sequencing was performed by the BGI-Shenzhen, Shenzhen, China. Extracted DNA was fragmented using a Covaris sonication system and sequencing libraries were prepared from 5 μg fragmented genomic DNA. End repair,  base addition and adaptor ligation steps were performed using Illumina’s Single-End DNA Sample Prep kit. Adaptor-ligated DNA was immunoprecipitated by anti-5mC using a commercial antibody (Diagenode), and MeDIP products were validated by quantitative PCR. MeDIP DNA was purified with ZYMO DNA Clean & Concentrator-5 columns, and amplified using adaptor-mediated PCR. DNA fragments between 220 and 320 bp in size were gel-excised, and amplification quality and quantity were evaluated by Agilent BioAnalyzer analysis. The libraries were subjected to highly parallel 50 bp single-end sequencing on the Illumina HiSeq platform.

E9.0-E9.5 wildtype and Nr6a1^−/−− embryos were isolated and yolk sac DNA genotyped in parallel. Total RNA was extracted from each whole embryo using TRIzol reagent and processed through an RNeasy column (QIAGEN) using the RNA clean-up protocol. Concentration and quality of RNA were determined by spectrophotometer and Agilent bioanalyzer analysis (Agilent Technologies, Inc., Palo Alto, CA). For array analysis, labelled mRNA targets were prepared from 150 ng total RNA using MessageAmp III RNA Amplification Kit (Applied Biosystems / Ambion, Austin, TX) according to manufacturer specifications. Array analysis was performed using Affymetrix GeneChip GeneChip Mouse Genome 430 2.0 Arrays processed with the GeneChip Fluidics Station 450 and scanned with a GeneChip Scanner 3000 7G using standard protocols.