Genome-wide DNA-methylation analysis of fat-1 transgenic bovine fetal fibroblasts and wild-type

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. Examine the genome-wide DNA-methylation in 2 different genotypes of bovine fetal fibroblasts.

研究目的：本研究发现，与野生型（wild-type）牛胎儿成纤维细胞相比，fat-1转基因牛胎儿成纤维细胞的能量代谢相关基因表达存在差异，因此需明确该表达差异是否由DNA甲基化（DNA methylation）改变所介导。
实验方法：采用德国杜塞尔多夫凯杰（Qiagen）公司的QIAamp Fast DNA Tissue试剂盒，按照制造商说明书提取总DNA；通过分光光度计检测A260/A280比值以定量DNA，当比值处于1.8~2.0区间时，判定DNA质量合格。将超声破碎后的DNA样品进行亚硫酸氢盐转化；使用美国密歇根州斯维夫特（Swift）公司的Accel-NGS Methyl-Seq DNA文库制备试剂盒，将接头连接至单链DNA片段。具体步骤如下：Adaptase反应为高效专有反应，可同时完成末端修复、3'端加尾以及首个截短接头互补序列与3'端的连接；延伸步骤通过引物延伸反应引入截短接头1；连接步骤仅将第二个截短接头连接至负链；索引PCR步骤可提升文库产量并引入全长接头。基于磁珠的SPRI纯化步骤用于去除寡核苷酸与小片段DNA，并更换酶促反应缓冲液体系。最终，依托LC Sciences公司的Illumina HiSeq 4000平台完成2×150bp双端测序。
实验结果：全基因组DNA甲基化分析显示，两组细胞间存在显著甲基化差异；KEGG富集分析表明，约80%的差异基因富集于代谢通路；且fat-1转基因组（FT）与野生型组（WT）牛胎儿成纤维细胞中，产热、脂肪酸延长、三羧酸（TCA）循环以及氧化磷酸化相关基因的DNA甲基化水平存在显著改变。
研究结论：fat-1转基因牛胎儿成纤维细胞与野生型细胞的全基因组DNA甲基化检测结果确实存在差异，且其中80%的差异与代谢过程相关。本研究针对两种不同基因型的牛胎儿成纤维细胞开展了全基因组DNA甲基化分析。