quantitative bisulphite pyrosequencing for opn4  (Pyrosequencing Inc)

Journal: Epigenetics & Chromatin

Article Title: Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

doi: 10.1186/s13072-020-00373-5

Figure Lengend Snippet: Altered cone response coupled with a decrease in OPN4 expression in the retina and the SCN of Afh/Afh mice. a Schematic representation of the RHT nodes, starting from the intrinsically photosensitive retinal ganglion cells (ipRGCs) to SCN and hypothalamus. b Flash electroretinogram (fERG) waveform of the cone response in Afh / Afh and +/+ animals ( n = 14 for each genotype) measured in photopic conditions. The saturated negative response and the positive response are represented with letters a and b. c Box plot showing the quantified amplitude values of the b wave between the two genotypes. d Box plot showing the quantified implicit response times after fERG stimulation. e Quantification of western blotting for unglycosylated (53 kDa) and glycosylated (85 kDa) OPN4 isoform protein levels in the two genotypes (+/+ n = 13, Afh/Afh n = 11). OPN4 levels were normalized to β-tubulin levels. f Opn4 expression levels in the SCN in Afh mutants (red) and controls (blue) ( n = 25 +/+ , n = 24 Afh/Afh ). g Relative expression of OPN4 normalized to multiple housekeeping genes at the two different time points, ZT0 and ZT12. The bars represent the average values for three different experiments ± SEMs (minimum n = 7 for each genotype at each time point). For each box plot (panels C and D), the small square indicates the mean, the central line illustrates the median, and the box limits indicate the 25th and 75th percentiles. The whiskers represent the 5th and 95th percentiles. * p < 0.05, ** p < 0.01, Student’s t test ( c – f ), two-way ANOVA plus Bonferroni post-test ( g ). All image analyses were performed with ImageJ software

Article Snippet: We performed quantitative bisulphite pyrosequencing for Opn4 , in Afh/Afh and +/+ SCNs collected at both ZT0 and ZT12.

Techniques: Expressing, Western Blot, Software

Journal: Epigenetics & Chromatin

Article Title: Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

doi: 10.1186/s13072-020-00373-5

Figure Lengend Snippet: Discrete and specific differences in the methylation profiles of Afh / Afh SCN vs +/+ animals. a Schematic representation of the Opn4 locus highlighting the CGI region (upper panel) at the beginning of the transcription start site (TSS, black arrow), as analyzed by pyrosequencing. Box plot of methylation values for each biological replicate averaged across all of the CpGs in the pyrosequencing amplicon (lower panel); the red lines represent the median, and the whiskers represent the 5th and 95th percentiles. b Pyrosequencing of Opn4 CGI methylation levels in Afh/Afh and +/+ mice at the two different time points, ZT0 and ZT12. c Principal component analysis of CGIs methylation vs time of day for +/+ and Afh/Afh animals after RRBS sequencing. d Logistic regression of the RRBS data identified 31 differentially methylated CpG islands (CGIs) (with a difference greater than 10%). Quantitation of methylation levels was performed with the SeqMonk software suite considering CGIs with a minimum count of 5 per position and at least 3 observed positions for each CGI across the dataset. The coloured scale on the right indicates the hypermethylated CGIs (in green) and the hypomethylated CGIs (in red). e Relative expression of a subset of genes with differentially methylated CGIs identified by the RRBS analysis at the two different time points, ZT0 and ZT12, normalized to the levels of multiple housekeeping genes. The bars represent the average values of three different experiments ± SEM. * p < 0.05, ** p < 0.01, two-way ANOVA plus Bonferroni post-test ( b ), Student’s t test ( a ), two-way ANOVA with Tukey’s post hoc test ( e ). a – b n = 5 for Afh/Afh and n = 6 for +/+ . c n = 6 for each genotype. d n = 3 for Afh/Afh and n = 3 for +/+ at each time point. e Minimum n = 7 per genotype at each time point

Article Snippet: We performed quantitative bisulphite pyrosequencing for Opn4 , in Afh/Afh and +/+ SCNs collected at both ZT0 and ZT12.

Techniques: Methylation, Amplification, Sequencing, Quantitation Assay, Software, Expressing