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Protocol publication

[…] oscape. The aligned sequencing data are available at the NCBI SRA archive as Bioproject PRJNA380108., DNA methylation changes associated with maternal HFD and offspring MCD challenge were assessed using RRBS, which involves sequencing of bisulfite-converted MspI fragment libraries. Liver genomic DNA was isolated using a combination of proteinase-K digestion and Purelink genomic DNA isolation kits (Life Technologies). Three biologically distinct pools of genomic DNA (containing n = 3 in each pool) were utilized to generate libraries. RRBS libraries were prepared as described by Gu et al [] with detailed methods provided in Supplementary methods. Reads were trimmed for adapter sequences using Trim Galore and filtered for quality score. Alignment and methylation calling were performed using Bismark. We first examined whether maternal HFD and offspring diets affected methylation of promoters and CpG islands (CGI). Promoters were sub-classified into those overlapping or devoid of CGIs. Frequency distribution of methylation status of features was computed. Statistical differences between groups were analyzed using χ2 test. Data analysis and summarization were done using SeqMonk and the DSS package in R []. Comparisons between different groups (CC, C-MCD, HC and H-MCD) were performed using Wald test, and P values were adjusted for multiple testing using the FDR method. CpGs with p < 0.0001 and a minimum difference in methylation (Δme) of 10% were retained. Differentially methylated regions (DMRs) were annotated with the closest/overlapping transcription start sites (TSSs) (±10 kb). The lists of differentially expressed genes were analyzed for GO biological process and molecular function enrichment using the BiNGO plugin in Cytoscape., Bacterial DNA was isolated from cecal contents using QIAamp Fast DNA stool mini kit (Qiagen) including a bead-beating step […]

Pipeline specifications

Software tools Trim Galore!, Bismark, SeqMonk