Journal: bioRxiv

Article Title: Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

doi: 10.1101/2019.12.20.885061

Figure Lengend Snippet: Principle of the EM-seq methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5-mC and 5-hmC, or BGT alone (right) to protect 5-hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C).

Article Snippet: For 5-mC detection, mouse brain genomic DNA (200 ng) was oxidized by incubating with 16 micrograms of TET2 (EM-seq TET2 Reaction Buffer (reconstituted), E7128A, E7131A diluted and E7130A) for 30 min at 37°C followed 30 min incubation with BGT (EM-seq component E7129A, NEB, Ip-swich, MA, USA) in the same buffer at 37°C.

Techniques: Amplification

Journal: bioRxiv

Article Title: Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

doi: 10.1101/2019.12.20.885061

Figure Lengend Snippet: Enzymatic deamination preserves the integrity of the DNA: a. qPCR results show the quantities of undamaged amplifiable DNA templates of different sizes after the enzymatic deamination (green) and bisulfite treatments (orange and blue). All quantifications are normalized to the values obtained for the enzymatic deamination experiments. b. Agilent 2100 Bioanalyzer trace on RNA 6000 pico chip comparing equal amounts of mouse E14 genomic DNA sheared to an average of 15 kb and treated with sodium bisulfite (green), BGT and APOBEC3A (red), or TET2 and APOBEC3A (blue) over the control ssDNA (magenta). Bisulfite treatment fragmented the DNA to an average of 800 bp, while enzymatically treated DNA show no notable size differences compared to control DNA. c. Agarose gel images of end-point PCR of six amplicons ranging from 388–4229 bp illustrating upper amplicon size limit for sodium bisulfite, TET2 and APOBEC3A, or BGT and APOBEC3A treated E14 genomic DNA. d. 731 bp amplicons from the agarose gels showed in (c) were cloned, sequenced and the methylation status determined for bisulfite (left panel), enzymatically converted for 5-mC (center panel) and 5-hmC (right panel) E14 genomic DNA. Open and closed circles indicate unmethylated and methylated, respectively.

Article Snippet: For 5-mC detection, mouse brain genomic DNA (200 ng) was oxidized by incubating with 16 micrograms of TET2 (EM-seq TET2 Reaction Buffer (reconstituted), E7128A, E7131A diluted and E7130A) for 30 min at 37°C followed 30 min incubation with BGT (EM-seq component E7129A, NEB, Ip-swich, MA, USA) in the same buffer at 37°C.

Techniques: Control, Agarose Gel Electrophoresis, Amplification, Clone Assay, Methylation