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Genome-wide analysis of DSBs sites in HEK293T cells

ABSTRACT: For genome-wide identification and analysis of endogenous DSBs in HEK293T cells chromosomes the ligation-mediated PCR and deep sequencing of the DSBs library were used. The cells were included in low-melt agarose, and the plugs were used for isolation of DNA in a solid phase using incubation in the solution containing 0.5 M EDTA (pH 9.5), 1% sodium laurylsarcosine, and 1-2 mg of proteinase K per ml for 40-48 hr at 50°C. Then DNA was isolated by electro-elution and used for ligation with biotinylated ds-adapter. To shorten large DNA molecules the digestion with Sau3A endonuclease was performed. Then the short regions of DNA attached to DSBs sites were selected using the streptavidin magnespere paramagnetic particles. To prepare samples for amplification of DSBs library the Sau3A adapter was ligated at the opposite to DSBs sites. PCR amplification was performed using the primers corresponding to biotinylated and Sau3A adapters. The library was used for paired end sequencing. The corresponding reads were processed and used for analysis of their distribution in human genome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE201829 | GEO | 2022/07/15

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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Genome-wide analysis of DSBs sites in HEK293T cells

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