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massarray epityper  (Epigenomics ag)

 
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    Epigenomics ag massarray epityper
    a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using <t>MassArray</t> and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Diagram of homology-directed repair (HDR) of the TET1 locus in TKO hESCs. Red letters indicate mutations of the WT sequence. The sequences of the two repaired lines (TKO-r1, TKO-r2) are shown below. d , Mass spectrometry analysis of 5hmC levels in WT, TET KO mutant lines, lines in which one allele of TET1 has been repaired (TKO-R) and lines which underwent HDR targeting but retained the TKO mutations in the TET1 locus (TKO-nr). For all mass spectrometry analysis, 2 mutant lines were used for all genotypes except for TKO. For TKO lines, 2 different passages of the same line were used for mass spectrometry measurements. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, *** P <0.001. e , Analysis of percent 5hmC at the PAX6 P0 promoter by Epimark, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. f , Top panel: Analysis of 5hmC peak at the PAX6 P0 promoter by hMe-Seal in WT hESCs. Bottom panel: Analysis of TET1 peak at the PAX6 P0 promoter by TET1 ChIP-Seq in WT hESCs. Shaded area represents the region of the PAX6 P0 promoter assayed for 5hmC by Epimark ( e ) and TET1 binding by ChIP-qPCR ( g ). g , ChIP-qPCR for TET1 in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01, *** P <0.001. h , Analysis of 5hmC and TET1 peaks at bivalent promoters in WT hESCs. The height above the x-axis reflects the normalized tag count. i , Percent DNA methylation change (TKO – WT) in bivalent promoters that have 5hmC peaks compared to bivalent promoters that don’t have 5hmC peaks.
    Massarray Epityper, supplied by Epigenomics ag, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/massarray+epityper+analysis/epityper+massarray/pmc05742051-256-0-8
    Average 90 stars, based on 1 article reviews
    massarray epityper - by Bioz Stars, 2026-10
    90/100 stars

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    1) Product Images from "TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells"

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

    Journal: Nature genetics

    doi: 10.1038/s41588-017-0002-y

    a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using MassArray and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Diagram of homology-directed repair (HDR) of the TET1 locus in TKO hESCs. Red letters indicate mutations of the WT sequence. The sequences of the two repaired lines (TKO-r1, TKO-r2) are shown below. d , Mass spectrometry analysis of 5hmC levels in WT, TET KO mutant lines, lines in which one allele of TET1 has been repaired (TKO-R) and lines which underwent HDR targeting but retained the TKO mutations in the TET1 locus (TKO-nr). For all mass spectrometry analysis, 2 mutant lines were used for all genotypes except for TKO. For TKO lines, 2 different passages of the same line were used for mass spectrometry measurements. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, *** P <0.001. e , Analysis of percent 5hmC at the PAX6 P0 promoter by Epimark, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. f , Top panel: Analysis of 5hmC peak at the PAX6 P0 promoter by hMe-Seal in WT hESCs. Bottom panel: Analysis of TET1 peak at the PAX6 P0 promoter by TET1 ChIP-Seq in WT hESCs. Shaded area represents the region of the PAX6 P0 promoter assayed for 5hmC by Epimark ( e ) and TET1 binding by ChIP-qPCR ( g ). g , ChIP-qPCR for TET1 in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01, *** P <0.001. h , Analysis of 5hmC and TET1 peaks at bivalent promoters in WT hESCs. The height above the x-axis reflects the normalized tag count. i , Percent DNA methylation change (TKO – WT) in bivalent promoters that have 5hmC peaks compared to bivalent promoters that don’t have 5hmC peaks.
    Figure Legend Snippet: a , Schematic for analysis of 5mC, 5hmC and TET1 binding at the PAX6 locus. Arrows represent the P0 and P1 promoter, the grey box represents the PAX6 mRNA transcript and the black box represents the PAX6 protein. The region analyzed for 5mC using MassArray and for 5hmC using hMe-Seal profiling is shown by a green box. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. (NE D4) Neuroectoderm differentiation day 4; (NE D10) Neuroectoderm Differentiation day 10. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Diagram of homology-directed repair (HDR) of the TET1 locus in TKO hESCs. Red letters indicate mutations of the WT sequence. The sequences of the two repaired lines (TKO-r1, TKO-r2) are shown below. d , Mass spectrometry analysis of 5hmC levels in WT, TET KO mutant lines, lines in which one allele of TET1 has been repaired (TKO-R) and lines which underwent HDR targeting but retained the TKO mutations in the TET1 locus (TKO-nr). For all mass spectrometry analysis, 2 mutant lines were used for all genotypes except for TKO. For TKO lines, 2 different passages of the same line were used for mass spectrometry measurements. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, *** P <0.001. e , Analysis of percent 5hmC at the PAX6 P0 promoter by Epimark, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. f , Top panel: Analysis of 5hmC peak at the PAX6 P0 promoter by hMe-Seal in WT hESCs. Bottom panel: Analysis of TET1 peak at the PAX6 P0 promoter by TET1 ChIP-Seq in WT hESCs. Shaded area represents the region of the PAX6 P0 promoter assayed for 5hmC by Epimark ( e ) and TET1 binding by ChIP-qPCR ( g ). g , ChIP-qPCR for TET1 in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01, *** P <0.001. h , Analysis of 5hmC and TET1 peaks at bivalent promoters in WT hESCs. The height above the x-axis reflects the normalized tag count. i , Percent DNA methylation change (TKO – WT) in bivalent promoters that have 5hmC peaks compared to bivalent promoters that don’t have 5hmC peaks.

    Techniques Used: Binding Assay, Sequencing, Mass Spectrometry, Mutagenesis, ChIP-sequencing, ChIP-qPCR, DNA Methylation Assay

    a , Schema for rescue of the NE differentiation defect in TKO hESCs using PAX6 overexpression or targeted demethylation of the PAX6 P0 promoter. b , qPCR analysis of neuroectoderm ( SOX1 ) and pluripotency ( OCT4 ) markers in WT and TKO cells without doxycycline (TKO) and with doxycycline treatment (TKO +PAX6) at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: one-way ANOVA, * P <0.05, ** P <0.01, *** P <0.001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at the endpoint of differentiation (D10) of TKO cells without doxycycline treatment (TKO) and with doxycycline treatment (TKO + PAX6). Scale bar indicates 100 µm. d , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut (left) or a dCas9-TET1CD (right) fusion protein. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. Methylation analysis at the PAX6 P0 promoter was analyzed for these cell lines with and without doxycycline treatment, n = 3 independent experiments. Statistical analysis: Student’s t test (two sided), **** P <0.0001. e , qPCR of PAX6 expression on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. PAX6 expression was analyzed for these cell lines with and without doxycycline treatment prior to differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), *** P <0.001. f , Immunofluorescence of PAX6 on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. TKO hESCs expressing the dCas9-TET1CD fusion and a non-targeting gRNA were also used as a control. PAX6 immunofluorscence was analyzed for these cell lines with and without doxycycline treatment prior to differentiation. Scale bar indicates 100 µm.
    Figure Legend Snippet: a , Schema for rescue of the NE differentiation defect in TKO hESCs using PAX6 overexpression or targeted demethylation of the PAX6 P0 promoter. b , qPCR analysis of neuroectoderm ( SOX1 ) and pluripotency ( OCT4 ) markers in WT and TKO cells without doxycycline (TKO) and with doxycycline treatment (TKO +PAX6) at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: one-way ANOVA, * P <0.05, ** P <0.01, *** P <0.001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at the endpoint of differentiation (D10) of TKO cells without doxycycline treatment (TKO) and with doxycycline treatment (TKO + PAX6). Scale bar indicates 100 µm. d , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut (left) or a dCas9-TET1CD (right) fusion protein. The location of each row of CpGs with respect to the P0 TSS is shown to the left of the heat map. Methylation analysis at the PAX6 P0 promoter was analyzed for these cell lines with and without doxycycline treatment, n = 3 independent experiments. Statistical analysis: Student’s t test (two sided), **** P <0.0001. e , qPCR of PAX6 expression on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. PAX6 expression was analyzed for these cell lines with and without doxycycline treatment prior to differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), *** P <0.001. f , Immunofluorescence of PAX6 on D10 of NE differentiation for TKO hESCs that express PAX6 targeting gRNAs with either a dCas9-TET1CD/Mut or a dCas9-TET1CD fusion protein. TKO hESCs expressing the dCas9-TET1CD fusion and a non-targeting gRNA were also used as a control. PAX6 immunofluorscence was analyzed for these cell lines with and without doxycycline treatment prior to differentiation. Scale bar indicates 100 µm.

    Techniques Used: Over Expression, Immunofluorescence, Methylation, Expressing, Control

    a , ChIP-qPCR for DNMT3B at the PAX6 locus in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter in WT, TKO and QKO hESCs. WT and TKO hESCs are passage-matched with the QKO hESCs. The location of each row of CpGs with respect to the TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. Quantification of the percent methylation is shown on the right, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at D10 of NE differentiation in WT, TKO and QKO cells. Scale bar indicates 100 µm. d , qPCR analysis of neuroectoderm ( PAX6 and SOX1 ) and pluripotency ( OCT4 and NANOG ) markers in WT, TKO and QKO cells at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001.
    Figure Legend Snippet: a , ChIP-qPCR for DNMT3B at the PAX6 locus in WT and TKO hESCs, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), * P <0.05, ** P <0.01. b , Heat map of MassArray analysis of 5mC at the PAX6 P0 promoter in WT, TKO and QKO hESCs. WT and TKO hESCs are passage-matched with the QKO hESCs. The location of each row of CpGs with respect to the TSS is shown to the left of the heat map. For each cell line three independent experiments are shown as three columns. Quantification of the percent methylation is shown on the right, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: Student’s t test (two sided), **** P <0.0001. c , Immunofluorescence of PAX6, SOX1 and OCT4 at D10 of NE differentiation in WT, TKO and QKO cells. Scale bar indicates 100 µm. d , qPCR analysis of neuroectoderm ( PAX6 and SOX1 ) and pluripotency ( OCT4 and NANOG ) markers in WT, TKO and QKO cells at D10 of NE differentiation, n = 3 independent experiments. Data presented are mean ± STD. Statistical analysis: black lines indicate comparisons to WT, one-way ANOVA, * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001.

    Techniques Used: ChIP-qPCR, Methylation, Immunofluorescence

    Related Articles

    Binding Assay:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Sequencing:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Mass Spectrometry:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Mutagenesis:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    ChIP-sequencing:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    ChIP-qPCR:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    DNA Methylation Assay:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Over Expression:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Immunofluorescence:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Methylation:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Expressing:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag

    Control:

    Article Title: Lifetime stress and war exposure timing may predict methylation changes at NR3C1 based on a pilot study in a warrior cohort in a small-scale society in Kenya.
    Article Snippet: was performed at Western Michigan University with the Gentra Puregene Blood Kit (Qiagen Inc.) using the DNA Purification from Body Fluid protocol. .. Genomic DNA (gDNA) samples were sent to the University of Michigan Epigenomics Core for EpiTYPER MassARRAY. .. All samples passed the core's QC.

    Article Title: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells
    Article Snippet: disaggregated using TrypLE and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. .. MassArray Epityper analysis was performed using the WCMC Epigenomics core. .. MassArray Epityper analysis consists of bisulfite treatment of genomic DNA, followed by PCR for the specific region of analysis, base specific cleavag



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    Epigenome-wide <t>DNA</t> <t>methylation</t> analysis reveals MIR-21 as the top hypermethylated region in CD4 T cells from FAE-treated patients. CD4 T cell DNA from 47 treatment-naïve, 35 FAE-treated and 16 GA-treated multiple sclerosis patients was analysed by using the Infinium MethylationEPIC BeadChip array. To identify differentially methylated regions between treated and untreated patients, we utilized a linear regression model at each individual CpG site to identify the contribution of treatment status in DNA methylation changes after controlling for age, gender, race, disease duration, the total CD4 T cell percentage as well as the percentage of CCR6−CCR4+, CCR6+CCR4+ and CCR6+CCR4− CD4 T cells in each sample. We then used a 1-kb sliding window to define genomic regions with closely located CpG sites, and then combined each CpG specific P-value from our previous linear regression model within a single region with the Stouffer’s method. This was followed by the Bonferroni correction for multiple hypothesis testing. DMRs were defined as regions with more than four CpG sites that have an absolute median β-value change > 0.02 and an adjusted combined P-value of < 0.01. Based on this analysis we uncovered 202 hypermethylated DMRs (containing 1545 CpGs) and only 13 hypomethylated DMRs (containing 158 CpGs) in FAE-treated patients compared to controls. (A and B) The CpGs distribution of these hypermethylated (hyperCpGs) and hypomethylated (hypoCpGs) DMRs was compared to that of the CpG distribution in the Illumina Infinium MethylationEPIC BeadChip (allCpGs). (C) Circos plot showing (from inside out); innermost first circle: chromosomal colors, numbers and size; second circle: a scatter plot with each point representing the genomic location and mean change in β-value of hypomethylated (in blue) and hypermethylated (in red) CpG sites; third circle: dark red perpendicular lines represent the genomic location of significantly hypermethylated DMRs; fourth circle: dark red scatter plot with each point representing the genomic location and mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs; outermost fifth circle: contains the names of genes whose promoters are located in these large DMRs (MIR-21 is shown in red). (D) Manhattan plot of the discovery cohort analysis showing mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs, where MIR-21 was the top differentially methylated locus. (E) Manhattan plot from the pair-wise longitudinal analysis showing that MIR-21 was the hypermethylated DMR with the most consistent and greatest methylation change in the validation cohort.
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    Epigenome-wide DNA methylation analysis reveals MIR-21 as the top hypermethylated region in CD4 T cells from FAE-treated patients. CD4 T cell DNA from 47 treatment-naïve, 35 FAE-treated and 16 GA-treated multiple sclerosis patients was analysed by using the Infinium MethylationEPIC BeadChip array. To identify differentially methylated regions between treated and untreated patients, we utilized a linear regression model at each individual CpG site to identify the contribution of treatment status in DNA methylation changes after controlling for age, gender, race, disease duration, the total CD4 T cell percentage as well as the percentage of CCR6−CCR4+, CCR6+CCR4+ and CCR6+CCR4− CD4 T cells in each sample. We then used a 1-kb sliding window to define genomic regions with closely located CpG sites, and then combined each CpG specific P-value from our previous linear regression model within a single region with the Stouffer’s method. This was followed by the Bonferroni correction for multiple hypothesis testing. DMRs were defined as regions with more than four CpG sites that have an absolute median β-value change > 0.02 and an adjusted combined P-value of < 0.01. Based on this analysis we uncovered 202 hypermethylated DMRs (containing 1545 CpGs) and only 13 hypomethylated DMRs (containing 158 CpGs) in FAE-treated patients compared to controls. (A and B) The CpGs distribution of these hypermethylated (hyperCpGs) and hypomethylated (hypoCpGs) DMRs was compared to that of the CpG distribution in the Illumina Infinium MethylationEPIC BeadChip (allCpGs). (C) Circos plot showing (from inside out); innermost first circle: chromosomal colors, numbers and size; second circle: a scatter plot with each point representing the genomic location and mean change in β-value of hypomethylated (in blue) and hypermethylated (in red) CpG sites; third circle: dark red perpendicular lines represent the genomic location of significantly hypermethylated DMRs; fourth circle: dark red scatter plot with each point representing the genomic location and mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs; outermost fifth circle: contains the names of genes whose promoters are located in these large DMRs (MIR-21 is shown in red). (D) Manhattan plot of the discovery cohort analysis showing mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs, where MIR-21 was the top differentially methylated locus. (E) Manhattan plot from the pair-wise longitudinal analysis showing that MIR-21 was the hypermethylated DMR with the most consistent and greatest methylation change in the validation cohort.

    Journal: Brain

    Article Title: Fumarates target the metabolic-epigenetic interplay of brain-homing T cells in multiple sclerosis

    doi: 10.1093/brain/awy344

    Figure Lengend Snippet: Epigenome-wide DNA methylation analysis reveals MIR-21 as the top hypermethylated region in CD4 T cells from FAE-treated patients. CD4 T cell DNA from 47 treatment-naïve, 35 FAE-treated and 16 GA-treated multiple sclerosis patients was analysed by using the Infinium MethylationEPIC BeadChip array. To identify differentially methylated regions between treated and untreated patients, we utilized a linear regression model at each individual CpG site to identify the contribution of treatment status in DNA methylation changes after controlling for age, gender, race, disease duration, the total CD4 T cell percentage as well as the percentage of CCR6−CCR4+, CCR6+CCR4+ and CCR6+CCR4− CD4 T cells in each sample. We then used a 1-kb sliding window to define genomic regions with closely located CpG sites, and then combined each CpG specific P-value from our previous linear regression model within a single region with the Stouffer’s method. This was followed by the Bonferroni correction for multiple hypothesis testing. DMRs were defined as regions with more than four CpG sites that have an absolute median β-value change > 0.02 and an adjusted combined P-value of < 0.01. Based on this analysis we uncovered 202 hypermethylated DMRs (containing 1545 CpGs) and only 13 hypomethylated DMRs (containing 158 CpGs) in FAE-treated patients compared to controls. (A and B) The CpGs distribution of these hypermethylated (hyperCpGs) and hypomethylated (hypoCpGs) DMRs was compared to that of the CpG distribution in the Illumina Infinium MethylationEPIC BeadChip (allCpGs). (C) Circos plot showing (from inside out); innermost first circle: chromosomal colors, numbers and size; second circle: a scatter plot with each point representing the genomic location and mean change in β-value of hypomethylated (in blue) and hypermethylated (in red) CpG sites; third circle: dark red perpendicular lines represent the genomic location of significantly hypermethylated DMRs; fourth circle: dark red scatter plot with each point representing the genomic location and mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs; outermost fifth circle: contains the names of genes whose promoters are located in these large DMRs (MIR-21 is shown in red). (D) Manhattan plot of the discovery cohort analysis showing mean beta change of significantly hypermethylated DMRs spanning more than 10 CpGs, where MIR-21 was the top differentially methylated locus. (E) Manhattan plot from the pair-wise longitudinal analysis showing that MIR-21 was the hypermethylated DMR with the most consistent and greatest methylation change in the validation cohort.

    Article Snippet: EpiTYPER MassArray® analysis DNA methylation analysis of ex vivo and in vitro stimulated cells was performed with EpiTYPERTM MassARRAY® system (Agena Bioscience) as previously described ( Moyon et al. , 2016 ) at the Epigenetics Core facility at the CUNY Advanced Science Research Center (ASRC).

    Techniques: DNA Methylation Assay, Methylation, Biomarker Discovery

    FAEs inhibit DNA demethylation at the MIR-21 promoter in a specific and dose-dependent manner. Naïve (CD45RO−CCR7+) and memory (CD45RO+) CD4 T cells were isolated from human PBMCs by FACS. The baseline DNA methylation levels of naïve (NaïveBL) and memory CD4 T cells (MemoryBL) at the (A) MIR-21 and (B) TNF promoters were measured ex vivo by EpiTYPER™ MassARRAY®. Naïve and memory CD4 T cells were also cultured with or without MMF at the specified concentration and stimulated with anti-CD3 and anti-CD28 coated beads for 3 days either without polarization or under Th17 polarizing conditions, after which DNA methylation levels at the (A) MIR-21 and (B) TNF promoters were measured by EpiTYPER™ MassARRAY®. (A) MMF was able to inhibit DNA demethylation of the MIR-21 promoter in naïve CD4 T cells, but not in memory CD4 T cells, in a dose dependent manner. (B) DNA methylation at the TNF promoter was not significantly changed by MMF in either naïve or memory CD4 T cells. (C) MMF had an even greater hypermethylating effect on the MIR-21 locus of naïve CD4 T cells that were stimulated under Th17 polarizing conditions. (D) MMF was also able to inhibit the demethylation of the MIR-21 promoter of naïve (CD45RO−CCR7+) CD8 T cells after 3 days of activation with anti-CD3 and anti-CD28 coated beads in vitro under Tc17 polarizing conditions. MMF20 = 20 µM of MMF; MMF50 = 50 µM of MMF; Veh = vehicle.

    Journal: Brain

    Article Title: Fumarates target the metabolic-epigenetic interplay of brain-homing T cells in multiple sclerosis

    doi: 10.1093/brain/awy344

    Figure Lengend Snippet: FAEs inhibit DNA demethylation at the MIR-21 promoter in a specific and dose-dependent manner. Naïve (CD45RO−CCR7+) and memory (CD45RO+) CD4 T cells were isolated from human PBMCs by FACS. The baseline DNA methylation levels of naïve (NaïveBL) and memory CD4 T cells (MemoryBL) at the (A) MIR-21 and (B) TNF promoters were measured ex vivo by EpiTYPER™ MassARRAY®. Naïve and memory CD4 T cells were also cultured with or without MMF at the specified concentration and stimulated with anti-CD3 and anti-CD28 coated beads for 3 days either without polarization or under Th17 polarizing conditions, after which DNA methylation levels at the (A) MIR-21 and (B) TNF promoters were measured by EpiTYPER™ MassARRAY®. (A) MMF was able to inhibit DNA demethylation of the MIR-21 promoter in naïve CD4 T cells, but not in memory CD4 T cells, in a dose dependent manner. (B) DNA methylation at the TNF promoter was not significantly changed by MMF in either naïve or memory CD4 T cells. (C) MMF had an even greater hypermethylating effect on the MIR-21 locus of naïve CD4 T cells that were stimulated under Th17 polarizing conditions. (D) MMF was also able to inhibit the demethylation of the MIR-21 promoter of naïve (CD45RO−CCR7+) CD8 T cells after 3 days of activation with anti-CD3 and anti-CD28 coated beads in vitro under Tc17 polarizing conditions. MMF20 = 20 µM of MMF; MMF50 = 50 µM of MMF; Veh = vehicle.

    Article Snippet: EpiTYPER MassArray® analysis DNA methylation analysis of ex vivo and in vitro stimulated cells was performed with EpiTYPERTM MassARRAY® system (Agena Bioscience) as previously described ( Moyon et al. , 2016 ) at the Epigenetics Core facility at the CUNY Advanced Science Research Center (ASRC).

    Techniques: Isolation, DNA Methylation Assay, Ex Vivo, Cell Culture, Concentration Assay, Activation Assay, In Vitro

    FAE therapy modifies the metabolic-epigenetic interplay in multiple sclerosis to reduce pathogenic CCR6+ CD4 and CD8 T cells. (A) CD4 T cells from treatment naïve patients with multiple sclerosis exhibit low DNA methylation levels at the MIR-21 locus and high expression of CCR6. CD8 T cells from treatment naïve multiple sclerosis patients also exhibit high levels of CCR6. (B) FAE therapy epigenetically modulates the MIR-21 locus in CD4 and CD8 T cells, which results in hypermethylation and downregulation of miR-21 expression in FAE-treated Th17 and Tc17 cells. This in turn allows for the upregulation of SMAD7 in both CD4 and CD8 T cells, a miR-21-5p and miR-21-3p target with inhibitory feedback on the TGF-b signalling pathway. Finally, FAEs reduce CCR6 expression and lower this potentially pathogenic brain-homing CCR6+ CD4 and CD8 T cell population in FAE-treated multiple sclerosis patients.

    Journal: Brain

    Article Title: Fumarates target the metabolic-epigenetic interplay of brain-homing T cells in multiple sclerosis

    doi: 10.1093/brain/awy344

    Figure Lengend Snippet: FAE therapy modifies the metabolic-epigenetic interplay in multiple sclerosis to reduce pathogenic CCR6+ CD4 and CD8 T cells. (A) CD4 T cells from treatment naïve patients with multiple sclerosis exhibit low DNA methylation levels at the MIR-21 locus and high expression of CCR6. CD8 T cells from treatment naïve multiple sclerosis patients also exhibit high levels of CCR6. (B) FAE therapy epigenetically modulates the MIR-21 locus in CD4 and CD8 T cells, which results in hypermethylation and downregulation of miR-21 expression in FAE-treated Th17 and Tc17 cells. This in turn allows for the upregulation of SMAD7 in both CD4 and CD8 T cells, a miR-21-5p and miR-21-3p target with inhibitory feedback on the TGF-b signalling pathway. Finally, FAEs reduce CCR6 expression and lower this potentially pathogenic brain-homing CCR6+ CD4 and CD8 T cell population in FAE-treated multiple sclerosis patients.

    Article Snippet: EpiTYPER MassArray® analysis DNA methylation analysis of ex vivo and in vitro stimulated cells was performed with EpiTYPERTM MassARRAY® system (Agena Bioscience) as previously described ( Moyon et al. , 2016 ) at the Epigenetics Core facility at the CUNY Advanced Science Research Center (ASRC).

    Techniques: DNA Methylation Assay, Expressing