RNA Extraction and Allele Quantification

Embryos were lysed in RLT buffer (Qiagen) supplemented with 0.01% 2-mercaptoethanol. After two rounds of vortexing (15 s each), lysates were applied directly to a QIAshredder spin column (Qiagen) and centrifuged for 3 min at >15,000 g. RNA was extracted using the RNAeasy Mini kit, including DNase treatment, and following the manufacturer's instructions. RNA samples were systematically run on an agarose gel to check their integrity. cDNA was prepared as described above for the gene expression analysis of mESCs, and was then PCR amplified with biotinylated primers and pyrosequenced for allele quantification on a Pyromark Q24 system (Qiagen). The same PCR approach was performed on no-reverse transcription control samples to confirm the absence of genomic DNA contamination. The primers used were designed with PyroMark Assay Design software and validated on XX polymorphic genomic DNA at a ratio of 50:50% (±4%). A list of primers and SNPs used for allele quantification can be found in Galupa et al. (2020) (link).