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Whole genome DNA methylation profiling of D2 medium spiny neurons in mouse nucleus accumbens using two independent library preparation methods

ABSTRACT: We isolated genomic DNA from adult male mouse nucleus accumbens (NAc) D2 medium spiny neurons (D2-MSN) by fluorescence-activated cell sorting. We performed the whole genome methylation profiling using two independent library preparation methods, namely the sodium bisulfite-based Accel-NGS Methyl-Seq (AM-seq) of Swift biosciences and the enzymatic conversion-based method Enzymatic Methyl-seq (EM-seq) from New England Biolabs, on two identical samples. We mapped about 400 million paired-end reads to the mouse genome (build mm10) and assessed measures of each methylome by two methods. This study provided a side-by-side comparison of two independent whole genome profiling methods for low-input neuronal samples.

ORGANISM(S): Mus musculus

PROVIDER: GSE195752 | GEO | 2022/03/04

REPOSITORIES: GEO

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Project description:Purpose:We found that fat-1 transgenic bovine fetal fibroblasts have changes in the expression of energy metabolism-related genes compared with wild-type, so whether this change is due to changes in DNA methylation needs to be determined. Methods:Total DNA was extracted using QIAamp Fast DNA Tissue Kit (Qiagen, Dusseldorf, Germany) following the manufacturer's procedure. The quantity of DNA was measured by reading A260/280 ratios by spectrophotometer. When A260/280 ratios located range 1.8 to 2.0, DNA was available. The fragmented DNA samples by using sonication were subjected to bisulfite conversion. The Accel-NGS Methyl-Seq DNA Library Kit (Swift, MI, USA) was utilized for attaching adapters to single-stranded DNA fragments. Briefly, the Adaptase step is a highly efficient, proprietary reaction that simultaneously performs end repair, tailing of 3 ends, and ligation of the first truncated adapter complement to 3 ends. The Extension step is used to incorporate truncated adapter 1 by a primer extension reaction. The Ligation step is used to add the second truncated adapter to the bottom strand only. The Indexing PCR step increases yield and incorporates full length adapters. Bead-based SPRI clean-ups are used to remove both oligonucleotides and small fragments, as well as to change enzymatic buffer composition. Finally, we performed the pair-end 2 150bp sequencing on an illumina Hiseq 4000 platform housed in the LC Sciences. Results:The results showed Genome-wide DNA-methylation analysis also showed differences between the two groups. KEGG analysis revealed that approximately 80% of the differential genes were enriched in metabolic pathways, and significant changes occurred in DNA methylation of genes related to thermogenesis, fatty acid elongation, TCA cycle and oxidative phosphorylation between FT and WT cells。 Conclusions：There are indeed differences between fat-1 transgenic bovine fetal fibroblasts and wild-type in genome-wide DNA methylation detection, and 80% of these differences are related to metabolism.

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Whole genome DNA methylation profiling of D2 medium spiny neurons in mouse nucleus accumbens using two independent library preparation methods

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