DNA Extractions and RRBS Data Analysis
Reduced Represented Bisulfite Sequencing (RRBS) Genomic DNA from F1 and F2 liver were isolated using the Zymo Quick-DNA Mini Prep Kit (Zymo Research Corp.). Upon submission, the gDNA samples were quantified using a fluorometric PicoGreen assay and analyzed for quality using the Nanodrop. All sampled passed the Input DNA Requirements outlines in Tecan’s Ovation RRBS Methyl-Seq System (Publication Number: M01394v7). gDNA samples were converted to sequencing libraries using  Tecan’s Ovation RRBS Methyl-Seq with TrueMethyl oxBS System following manufacturers protocols (Part # 9522-A01). In summary, extracted gDNA was digested by MspI and then ligated to indexed sequencing adapters. Libraries were bisulfite converted (oxBS module was not used) and amplified. Final library size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen). Libraries were pooled and sequenced on one lane of an S4 2x150 paired-end run on the Illumina NovaSeq 6000 System.
R (R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.) and Linux command line tools were used for RRBS analysis. FastQC (version11.3) assessed overall sequencing quality, and TrimGalore(F. Krueger 2015) (version 4.5) with default settings trimmed low-quality bases, adapter sequences, and end-repair bases from 3’ ends. Bismark(Felix Krueger and Andrews 2011) (version 19.0) aligned and called methylation. Reads were aligned to mm10 mouse reference genome using Bowtie2(Langmead and Salzberg 2012) (version 2.3.4) using default parameters. Methylation calls were reported for all nucleotides with the minimum read depth of 5. Paired raw fastq.gz reads were uploaded to the Sequence Read Arcive and can be accesed under Bioproject Number (PRJNA881737) . The DSS package in R (version 2.32.0) detected differential methylation in liver for F1 and F2 females versus sex- and generation- matched 1ppb controls (n = 3 per group) for sites with a minimum coverage of 10 reads. The DMLtest and callDML functions were used to detect differentially methylated CpGs (DMCs). Smoothing was set to FALSE in DMLtest as is recommended for sparse data. Differentially methylated regions (DMRs) were identified with callDMR. The p-value threshold was set to FDR < 0.001 for both tests with delta > 0.01 required to call DMCs. A delta value was not enforced for DMRs; all CpGs within a potential DMR need not exhibit the same direction of change, meaning a delta threshold could lead to false negatives. The Annotatr R package was used to annotate DMCs and DMRs to CpG islands, genes, and intergenic regions in the mm10 genome(Cavalcante and Sartor 2016). The functions plot_annotation and plot_categorical functions were used to generate figures.