Protocol matters – reproducibility and rigor of DNA methylation data sets [RNA-seq]

While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Indeed, separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Genome-wide DNA methylation analysis of hippocampal tissues from wild-type rats across three independent laboratories revealed that seemingly minor protocol differences resulted in significant epigenome profile changes, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results. Overall design: SAS Sprague Dawley (SD) rats were purchased from the nearest vendors (Charles River and Envigo) from our three project sites, including Legacy Research (Site #1), Trinity College (Site #2), and the University of Alabama at Birmingham (Site #3). We identified factors that are typically easy to match, factors that may not always be considered, and factors that are challenging to match. A total of 27 factors were collected in four major areas including “before-each-laboratory”, “at-each-laboratory”, “up-to-sacrifice”, and “dissection”. Rats were 8.0 to 8.3 weeks of age at the time of arrival from the vendors. Entire hippocampi (from both hemispheres) were harvested from rats (n=5~6) 18 to 27 days after arrival. Whole hippocampi from each animal were surgically dissected and flash-frozen in liquid N2 and stored at -80°C before being shipped to the University of North Dakota (UND), where samples were collected, de-identified, and stored at -80°C for deep sequencing analysis. Once collected, all samples were processed using Qiagen's AllPrep DNA/RNA Mini Kit to individually extract RNA and DNA from each flash-frozen sample. All RNA and DNA samples were stored at -80°C before being sent frozen in dry ice to the University of Michigan Genomics and Epigenomics Core for RNA-Seq and reduced representation bisulfite sequencing (RRBS). Differentially expressed genes (DEGs) and differentially methylated CpG sites/Genes (DMCs/DMGs) were identified between each pair of project sites and examined for enriched biological functions and pathways.