pyrosequencing-based dna methylation analysis  (Pyrosequencing Inc)

Journal: PLoS ONE

Article Title: DNA Methylation Dynamics in Blood after Hematopoietic Cell Transplant

doi: 10.1371/journal.pone.0056931

Figure Lengend Snippet: (A) Diagram of the experimental design. The differences of the global methylation levels between donors, pre-HCT recipients and post-HCT recipients were assessed by a pyrosequencing based methylation assay of repetitive DNA elements (LINE1 and NBL2) in whole blood. (B) NBL2 ΔMet values between donors, pre-HCT recipients, and 1 month post-HCT recipients. (C) NBL2 ΔMet mean values between donors and recipients up to 12 months post-transplant. The dotted line marks the ΔMet mean value between donors and 1 month post-HCT recipients (ΔMet = 5.8450). During the follow up of the transplant, the mean values barely deviated from the initial post-HCT ΔMet. (D) LINE1 ΔMet values between donors, pre-HCT recipients and 1 month post-HCT recipients. (E) LINE1 ΔMet mean values between donors and recipients up to 12 months post-transplant. The dotted line marked the ΔMet mean value between donors and 1 month post-HCT recipients (ΔMet = 5.383).

Article Snippet: Pyrosequencing-based DNA methylation analysis is relatively inexpensive, is very sensitive, and provides an interesting alternative to protein and mRNA detection in blood samples, allowing the development of novel biomarkers in a large array of human diseases.

Techniques: Methylation

Journal: PLoS ONE

Article Title: DNA Methylation Dynamics in Blood after Hematopoietic Cell Transplant

doi: 10.1371/journal.pone.0056931

Figure Lengend Snippet: Scatter plots showing DNA methylation in donors versus post-transplant recipients 1 month and 6 months post-HCT. Red dots represent CpG sites with methylation values altered more than 20% relative to donor values.

Article Snippet: Pyrosequencing-based DNA methylation analysis is relatively inexpensive, is very sensitive, and provides an interesting alternative to protein and mRNA detection in blood samples, allowing the development of novel biomarkers in a large array of human diseases.

Techniques: DNA Methylation Assay, Methylation

Journal: Genome Biology

Article Title: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: Over-expression of Klhl13 and Dusp9 in male mESCs leads to an enhanced pluripotency state and slower differentiation kinetics. a Schematic representation of the dCas9-SunTag system used for gene activation. b–e To over-express Dusp9 (yellow) and Klhl13 (blue), male E14 mESCs, stably expressing the doxycycline-inducible SunTag system, were either transduced with one of two different sgRNAs targeting the respective promoter regions or with non-targeting control (NT) sgRNAs and were treated for 3 days with 1 μg/ml doxycycline as indicated. Protein levels of Dusp9 (left) and Klhl13 (right) were quantified via immunoblotting ( b ), expression levels of MAPK target genes Spry4 and Egr1 ( c ) and of naive pluripotency factors Nanog and Prdm14 ( e ) were assessed by qPCR and phosphorylation of Mek and Erk was quantified by immunoblotting ( d ). The immunoblot signals were normalized to Tubulin ( b ) or to total Mek/Erk ( d ) and to the mean of two doxycycline-treated non-targeting control sgRNAs. qPCR measurements were normalized to two housekeeping genes and to the respective untreated control (−Dox). Dots and triangles depict individual measurements of the two different sgRNAs, and thick bars show the mean of three biological replicates. f Dusp9- and Klhl13 over-expressing mESCs were treated with 1 μg/ml doxycycline 24 h before differentiation via LIF withdrawal for 4 days, and expression levels of pluripotency factors were measured by qPCR at different time points as indicated. Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA). * p < 0.05 in a two-tailed paired Student’s t test comparing the Dusp9/Klhl13 over-expressing samples and the non-targeting controls (mean of sgRNA1 and sgRNA2)

Article Snippet: Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test

Journal: Genome Biology

Article Title: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: Over-expression of Klhl13 and Dusp9 in male mESCs leads to an enhanced pluripotency state and slower differentiation kinetics. a Schematic representation of the dCas9-SunTag system used for gene activation. b–e To over-express Dusp9 (yellow) and Klhl13 (blue), male E14 mESCs, stably expressing the doxycycline-inducible SunTag system, were either transduced with one of two different sgRNAs targeting the respective promoter regions or with non-targeting control (NT) sgRNAs and were treated for 3 days with 1 μg/ml doxycycline as indicated. Protein levels of Dusp9 (left) and Klhl13 (right) were quantified via immunoblotting ( b ), expression levels of MAPK target genes Spry4 and Egr1 ( c ) and of naive pluripotency factors Nanog and Prdm14 ( e ) were assessed by qPCR and phosphorylation of Mek and Erk was quantified by immunoblotting ( d ). The immunoblot signals were normalized to Tubulin ( b ) or to total Mek/Erk ( d ) and to the mean of two doxycycline-treated non-targeting control sgRNAs. qPCR measurements were normalized to two housekeeping genes and to the respective untreated control (−Dox). Dots and triangles depict individual measurements of the two different sgRNAs, and thick bars show the mean of three biological replicates. f Dusp9- and Klhl13 over-expressing mESCs were treated with 1 μg/ml doxycycline 24 h before differentiation via LIF withdrawal for 4 days, and expression levels of pluripotency factors were measured by qPCR at different time points as indicated. Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA). * p < 0.05 in a two-tailed paired Student’s t test comparing the Dusp9/Klhl13 over-expressing samples and the non-targeting controls (mean of sgRNA1 and sgRNA2)

Article Snippet: Since Dusp9 has been suggested to be responsible for the reduction of global CpG methylation levels typically observed in female mESCs (20–30% compared to 60–80% in male mESCs) [ , , ], we analyzed how over-expression of Dusp9 and Klhl13 affected global DNA methylation through the pyrosequencing-based luminometric DNA methylation assay (LUMA; Fig. g).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: DNA methylation and hydroxymethylation analyses of the active LINE-1 subfamilies in mice

doi: 10.1038/s41598-017-14165-7

Figure Lengend Snippet: Pyrosequencing-based DNA methylation assay of the active LINE-1 subfamilies in mice. ( a ) Schematic representation of 5′-UTR of the active LINE-1 (TfI, A, and GfII) in mice, drawn from previous studies , . Designed primers and examined CpG sites are indicated. 5′-UTR of the active LINE-1 subfamilies generally consists of a variable number of monomers and truncated monomers as well as a nonmonomer region upstream of ORF1. Definitions of monomers and nonmonomers were determined previously , . Numbers in brackets indicate the total copy number of full length elements, and rough age of the subfamily . Myr, million years; ORF, open reading frame; F, forward primer; R, reverse primer; S, sequence primer. ( b ) Alignment of the nonmonomer sequences of TfI, A, and GfII. Nonconserved sequences among these subfamilies are highlighted in black. The analyzed CpGs within the nonmonomer sequences (TfI_CpG#1, TfI_CpG#2, A, and GfII_CpG#3) are boxed. Note that GfII_CpG#1 and GfII_CpG#2 are located in the truncated monomer sequence. ( c ) Agarose gel electrophoresis analysis of the PCR amplicons. The expected amplicon sizes of TfI, A, and GfII are 261 bp, 129 bp, and 244 bp, respectively. A 100-bp DNA ladder (TakaraBio) was used. Arrows indicate respective sizes of bands. ( d ) A representative pyrogram in pyrosequencing analysis (TfI_CpG#2). The pyrosequencing reaction starts with an input of enzyme (E) followed by substrates (S). The sequence after S is as listed as dispensation order in Table . The shaded site is the cytosine analyzed in this study.

Article Snippet: Figure 1 Pyrosequencing-based DNA methylation assay of the active LINE-1 subfamilies in mice. ( a ) Schematic representation of 5′-UTR of the active LINE-1 (TfI, A, and GfII) in mice, drawn from previous studies , .

Techniques: DNA Methylation Assay, Sequencing, Agarose Gel Electrophoresis, Amplification

Journal: Scientific Reports

Article Title: DNA methylation and hydroxymethylation analyses of the active LINE-1 subfamilies in mice

doi: 10.1038/s41598-017-14165-7

Figure Lengend Snippet: mC and hmC levels of the active LINE-1 subfamilies in the adult mouse brain regions. For TfI, two CpG sites located within the nonmonomer region; for A, one CpG site located within the nonmonomer region; and for GfII, three CpG sites (two located within the truncated monomer, and one within the nonmonomer region) were analyzed. Three samples were available for analysis for each CpG site, except for the samples indicated by # symbol, for which only two were available. Values are given as mean ± standard deviation. mC, DNA methylation; hmC, hydroxymethylation; FC, frontal cortex; Hp, hippocampus; Cb, cerebellum; BG, basal ganglia.

Article Snippet: Figure 1 Pyrosequencing-based DNA methylation assay of the active LINE-1 subfamilies in mice. ( a ) Schematic representation of 5′-UTR of the active LINE-1 (TfI, A, and GfII) in mice, drawn from previous studies , .

Techniques: Standard Deviation, DNA Methylation Assay

Journal: Scientific Reports

Article Title: DNA methylation and hydroxymethylation analyses of the active LINE-1 subfamilies in mice

doi: 10.1038/s41598-017-14165-7

Figure Lengend Snippet: mC and hmC levels of the active LINE-1 subfamilies in the brain and nonbrain tissues. The data from four brain regions were averaged and treated as a single tissue, Brain (AVE). Three samples were available for analysis for each CpG site, except for the samples indicated by # symbol, for which only two were available. Values are given as mean ± standard deviation. *Statistically significant (ANOVA followed by Tukey’s test, P < 0.05). mC, DNA methylation; hmC, hydroxymethylation.

Article Snippet: Figure 1 Pyrosequencing-based DNA methylation assay of the active LINE-1 subfamilies in mice. ( a ) Schematic representation of 5′-UTR of the active LINE-1 (TfI, A, and GfII) in mice, drawn from previous studies , .

Techniques: Standard Deviation, DNA Methylation Assay

Journal: Scientific Reports

Article Title: DNA methylation and hydroxymethylation analyses of the active LINE-1 subfamilies in mice

doi: 10.1038/s41598-017-14165-7

Figure Lengend Snippet: Correlation of actual mC levels between different two CpG sites. Pairs of ( a ) GfII_CpG#1 and GfII_CpG#2, and ( b ) A and GfII_CpG#3 were the only ones that showed significant correlations ( P < 0.05 after Bonferroni correction). Each symbol represents data from one individual tissue ( N = 12 for brain regions and N = 3 each for other tissues, except for the ovary tissue in GfII_CpG#3, for which only two were available). Symbols with * on the right corner indicate that two samples are overlapped on each other in the graph, because they showed the same DNA methylation levels.

Article Snippet: Figure 1 Pyrosequencing-based DNA methylation assay of the active LINE-1 subfamilies in mice. ( a ) Schematic representation of 5′-UTR of the active LINE-1 (TfI, A, and GfII) in mice, drawn from previous studies , .

Techniques: DNA Methylation Assay