Journal: Frontiers in Epigenetics and Epigenomics

Article Title: Benchmark of the Oxford Nanopore, EM-seq, and HumanMethylationEPIC BeadChip for the detection of the 5mC sites in cancer and normal samples

doi: 10.3389/freae.2024.1362926

Figure Lengend Snippet: Summary of the comparison of characteristics and performance of DNA methylation detection methods based on literature search and obtained benchmarking results.

Article Snippet: Bisulfite conversion was performed using the DNA Zymo Methylation Kit (Zymo Research, United States) for 1,000 ng of genomic DNA.

Techniques: Comparison, DNA Methylation Assay, Dispersion

Journal: bioRxiv

Article Title: The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis

doi: 10.1101/2022.12.27.522067

Figure Lengend Snippet: (A) Upper panel: Map of MFCS1-luc2 used for assays in (B). Lower panel: Alignment of MFCS1 highly conserved sequences. Nucleotides identical in more than two sequences are shaded in grey. Conserved Hox-binding motifs are boxed. A light bule line underlies the 14-thymidine repeat in X. tropicalis (See Materials and Methods for details.). (B) Firefly luciferase activity of MFCS1-luc2 using a minimum promoter (minP) or shh promoter (shh pro) was normalized to Renilla luciferase control activity. The mean value of mock samples by the control construct with the minimum promoter was adjusted to 1.0. Error bars, standard deviation from the mean (n=3). *Significant increase from mock (p<0.01). **Significant increase in Fgf2+Hoxd13, Fgf4+Hoxd13 and Fgf8+Hoxd13 from sole Fgf2, Fgf4 and Fgf8, respectively (p<0.01). (C) Methylation pattern of endogenous and transgene MFCS1 (line 1). Samples were obtained from the autopod (pink). Each circle corresponds to a CpG site (see ). Lower panel: Increases in average methylation rate of the MFCS1 sequences after metamorphosis. (D) Model for epigenetic silencing of gene expression through DNA methylation of MFCS1. Hoxd13 activates hypomethylated MFCS1 and results in transcription of a gene under control of MFCS1 in the tadpole limb (left). However, Hoxd13 is unable to activate highly methylated MFCS1 in a froglet limb and results in inactive transcription of the gene (right).

Article Snippet: The genomic DNA was treated with sodium bisulfite using an EZ DNA Methylation-Gold Kit following the protocol of the manufacturer (Zymo Research).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Methylation, Expressing, DNA Methylation Assay

Journal: bioRxiv

Article Title: The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis

doi: 10.1101/2022.12.27.522067

Figure Lengend Snippet: (A) Methylation-sensitive restriction digestion visualized by agarose gel electrophoresis. Plasmid DNA of the MFCS1- shh promoter- luc2 reporter was treated with CpG methyltransferase (M. SssI, NEB) according to the manufacturer’s protocol. Methylation efficiency of the plasmid DNA was evaluated by digestion with methylation-sensitive (HpaII) and -insensitive (MspI) restriction enzymes. The CpG methylation prohibited the digestion of the plasmid DNA specifically with HpaII. M, l/HindIII marker. (B) Luciferase activity in NIH3T3 cells transfected with the methylated or non-treated MFCS1-luc2 reporter constructs and the Hoxd13 expression construct. The mean value of luciferase activity in cells transfected with the non-treated reporter and mock expression constructs was set to 1.0. Error bars: standard deviation from the mean (n=3). While a significant increase of Hoxd13 -induced luciferase activity was observed in cells transfected with the unmethylated reporter construct (*p<0.01), being consistent with , there was no significant increase in cells transfected with the methylated reporter construct despite Hoxd13 expression. N. S., not significant.

Article Snippet: The genomic DNA was treated with sodium bisulfite using an EZ DNA Methylation-Gold Kit following the protocol of the manufacturer (Zymo Research).

Techniques: Methylation, Agarose Gel Electrophoresis, Plasmid Preparation, CpG Methylation Assay, Marker, Luciferase, Activity Assay, Transfection, Construct, Expressing, Standard Deviation