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| Status |
Public on Jan 12, 2022 |
| Title |
Protocol matters – reproducibility and rigor of DNA methylation data sets [BiSulfite-seq] |
| Organism |
Rattus norvegicus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
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.
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| 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.
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| Contributor(s) |
Hur J, Boison D, Lubin FD, Geiger JD, Masino SA, Guo K |
| Citation(s) |
35013473 |
| Submission date |
Jan 14, 2021 |
| Last update date |
Jan 20, 2022 |
| Contact name |
Junguk Hur |
| E-mail(s) |
junguk.hur@med.und.edu
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| Organization name |
University of North Dakota
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| Department |
Biomedical Sciences
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| Street address |
1301 N. Columbia Rd. Stop 9037
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| City |
Grand Forks |
| State/province |
ND |
| ZIP/Postal code |
58202-9037 |
| Country |
USA |
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| Platforms (1) |
| GPL22396 |
Illumina HiSeq 4000 (Rattus norvegicus) |
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| Samples (17)
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| This SubSeries is part of SuperSeries: |
| GSE164833 |
Protocol matters – reproducibility and rigor of DNA methylation data sets |
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| Relations |
| BioProject |
PRJNA692180 |
| SRA |
SRP301758 |
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