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. 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.