Computational protocol: Paternal heat exposure causes DNA methylation and gene expression changes of Stat3 in Wild guinea pig sons

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Protocol publication

[…] Primers (Table ) were designed based on the C. aperea whole genome sequence (Weyrich et al. ) using the Geneious® software. For maximum quantification accuracy, primers producing amplicons of 120–200 bp were chosen with an optimal annealing temperature (T a) of 55°C and a length of 20+/− 2 nucleotides. Theoretical T a values were calculated with the CFX Manager Software (Bio‐Rad GmbH, Munich, Germany). The cDNA template for PCR testing was freshly synthesized from randomly selected samples. Negative controls were set up for each primer pair. A positive PCR control was prepared using the Stat3 primer pair. For visualization of PCR products, a 1.5% agarose gel was prepared (1.5 g agarose GOLD® (Peqlab) + 100 mL 1x TBE buffer, 3 μL GelRed™ (Biotium, Cologne, Germany)). 5 μL PCR product was added to 1 μL loading dye (6x orange DNA loading Dye; Fermentas, St. Leon‐Rot, Germany). The 50‐bp ladder (O'GeneRuler™ 0.1 μg/μL, Thermo Scientific) was used for size determination. PCR reaction conditions for primer pairs were optimized. Amplicons were verified in size using agarose gel electrophoresis as well as by Sanger sequencing (Sanger et al. ) using the BigDye® Terminator v3.1 Cycle Sequencing Kit and BigDye® XTerminator™ Purification Kit (Life Technologies, Darmstadt, Germany). Capillary electrophoresis was performed using the ABI Prism 3130xl Genetic Analyzer and associated Collection software v. 3.0 (Life Technologies). Sequences were analyzed using the Sequence Analyser® v. 5.2 (Life Technologies) and BioEdit v. 7.2.5 software (Hall 1999). NCBI BLAST search finally confirmed sequence specificity. [...] We applied Fisher's exact test for methylation levels and a paired Wilcoxon signed‐rank test to test for significant differences between F1C and F1H for normalized expression values using R v.3.1.1 (http://cran.r-project.org/bin/windows/base/). A paired Wilcoxon signed‐rank test was also applied to investigate a potential maternal effect; wherefore, we first averaged the expression values of the sons per father–mother combination (n = 10). The plots were generated in R with ggplot2 v1.0.0 and are displayed as whisker plots with 1.5*IQR (http://cran.r-project.org/web/packages/ggplot2/citation.html). Normalized expression values were also tested for significant associations with specific methylation changes using Pearson's test in R. […]

Pipeline specifications

Software tools Geneious, CFX Manager, BioEdit, BLASTN, Ggplot2
Applications Miscellaneous, WGS analysis
Organisms Cavia porcellus, Homo sapiens