cpg methyltransferase m sssi  (New England Biolabs)


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    Name:
    CpG Methyltransferase M SssI
    Description:
    CpG Methyltransferase M SssI 500 units
    Catalog Number:
    M0226L
    Price:
    288
    Size:
    500 units
    Category:
    DNA Methylases
    Score:
    85
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    New England Biolabs cpg methyltransferase m sssi
    CpG Methyltransferase M SssI
    CpG Methyltransferase M SssI 500 units
    https://www.bioz.com/result/cpg methyltransferase m sssi/product/New England Biolabs
    Average 99 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    cpg methyltransferase m sssi - by Bioz Stars, 2019-12
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    Images

    1) Product Images from "Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor"

    Article Title: Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025922

    Evaluation of predicted ZREs in three EBV promoters. A. Summary of the known (filled box) and predicted (open box) core ZRE sequences within the proximal 500 nucleotides of the indicated BZLF1 , BRLF1 and BMRF1 promoters. The arrows represent the transcription start sites. Stars represent CpG ZREs. B. Double strand oligonucleotides were generated with the core ZRE sequence and at least 10 nucleotides of cognate sequence on either side. Following radio labeling, these were incubated with in vitro translated Zta and subject to EMSA. The reactions contained no protein, 0, control lysate, C or Zta, Z. The DNA probes are indicated above with their originating promoters. C. Double strand oligonucleotides were generated with the core ZRE sequence and 10 nucleotides of cognate sequence on either side. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
    Figure Legend Snippet: Evaluation of predicted ZREs in three EBV promoters. A. Summary of the known (filled box) and predicted (open box) core ZRE sequences within the proximal 500 nucleotides of the indicated BZLF1 , BRLF1 and BMRF1 promoters. The arrows represent the transcription start sites. Stars represent CpG ZREs. B. Double strand oligonucleotides were generated with the core ZRE sequence and at least 10 nucleotides of cognate sequence on either side. Following radio labeling, these were incubated with in vitro translated Zta and subject to EMSA. The reactions contained no protein, 0, control lysate, C or Zta, Z. The DNA probes are indicated above with their originating promoters. C. Double strand oligonucleotides were generated with the core ZRE sequence and 10 nucleotides of cognate sequence on either side. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.

    Techniques Used: Generated, Sequencing, Labeling, Incubation, In Vitro, Methylation

    Zta recognition and methylation dependence of PFM CpG5 predicted CpG containing ZREs. A. Flow diagram illustrating the information flow from the PFM to the predictions of novel ZREs in the EBV genome and their subsequent evaluation. B. Core heptamer sequences, in both forward and reverse complement, of PFM CpG5 predicted CpG containing ZREs within the EBV genome. C. PFM CpG5 was used to predict the potential for further ZREs in the EBV genome. Double strand oligonucleotides were generated. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
    Figure Legend Snippet: Zta recognition and methylation dependence of PFM CpG5 predicted CpG containing ZREs. A. Flow diagram illustrating the information flow from the PFM to the predictions of novel ZREs in the EBV genome and their subsequent evaluation. B. Core heptamer sequences, in both forward and reverse complement, of PFM CpG5 predicted CpG containing ZREs within the EBV genome. C. PFM CpG5 was used to predict the potential for further ZREs in the EBV genome. Double strand oligonucleotides were generated. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.

    Techniques Used: Methylation, Flow Cytometry, Generated, Labeling, In Vitro, Incubation

    2) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3073

    MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
    Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P

    Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification

    3) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3073

    MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
    Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P

    Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification

    4) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

    Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096512

    Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
    Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p

    Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    5) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"

    Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx900

    Determination of de novo and maintenance CpG methylation in Tnp1 and Pfn3 minigenes with distinct splicing capacities. ( A ) Schematic representation of regions interrogated by bisulfite-pyrosequencing (BS-pyroseq) in the Tnp1 and Pfn3 minigenes. ( B ) Per-base heatmap for percent CpG methylation at the 7 interrogated sites within Tnp1 exon 2. Values represent averages from duplicate BS-pyroseq performed on biological replicates of Dox-induced Tnp1 host cells transfected with vector encoding DNMT3A, DNMT3B or control (left). Average methylation across the seven sites in DNMT3-transfected and control cells (right), mean ± SEM. ( C ) BS-pyroseq of the 18 CpGs overlapping the Pfn3 ORF, as in (A). P -values = two-tailed Student's t -test comparing average methylation DNMT3-transfected cells versus control, as indicated. ( D ) CpG-free Tnp1 expression vector was methylated in vitro using M.SssI and stably transfected into 293T cells for random integration. ( E ) MSRE-qPCR for relative Tnp1 DNA methylation from all stable gDNA and ΔSS minigene clones at 21, 49 and 77 days post-transfection summarized in boxplot; white diamonds indicate average Tnp1 methylation from seven and six independent Tnp1 gDNA and ΔSS clones, respectively. Student's t -test comparing mean methylation between indicated pairs yielded no significant differences (NS).
    Figure Legend Snippet: Determination of de novo and maintenance CpG methylation in Tnp1 and Pfn3 minigenes with distinct splicing capacities. ( A ) Schematic representation of regions interrogated by bisulfite-pyrosequencing (BS-pyroseq) in the Tnp1 and Pfn3 minigenes. ( B ) Per-base heatmap for percent CpG methylation at the 7 interrogated sites within Tnp1 exon 2. Values represent averages from duplicate BS-pyroseq performed on biological replicates of Dox-induced Tnp1 host cells transfected with vector encoding DNMT3A, DNMT3B or control (left). Average methylation across the seven sites in DNMT3-transfected and control cells (right), mean ± SEM. ( C ) BS-pyroseq of the 18 CpGs overlapping the Pfn3 ORF, as in (A). P -values = two-tailed Student's t -test comparing average methylation DNMT3-transfected cells versus control, as indicated. ( D ) CpG-free Tnp1 expression vector was methylated in vitro using M.SssI and stably transfected into 293T cells for random integration. ( E ) MSRE-qPCR for relative Tnp1 DNA methylation from all stable gDNA and ΔSS minigene clones at 21, 49 and 77 days post-transfection summarized in boxplot; white diamonds indicate average Tnp1 methylation from seven and six independent Tnp1 gDNA and ΔSS clones, respectively. Student's t -test comparing mean methylation between indicated pairs yielded no significant differences (NS).

    Techniques Used: CpG Methylation Assay, Transfection, Plasmid Preparation, Methylation, Two Tailed Test, Expressing, In Vitro, Stable Transfection, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Clone Assay

    6) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2010.01124.x

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
    Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    7) Product Images from "The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes"

    Article Title: The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000935

    Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.
    Figure Legend Snippet: Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.

    Techniques Used: Methylated DNA Immunoprecipitation, CpG Methylation Assay, Infection, Methylation, Isolation, Immunoprecipitation, Hybridization, Fluorescence, Generated, Positive Control, In Vitro, Negative Control

    8) Product Images from "Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing"

    Article Title: Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing

    Journal:

    doi: 10.1088/1478-3975/9/6/065001

    The 2905 bp DNA template (A) showing Ava1 restriction sites. The sequence contains 951 cytosines (32.2% C content) and 259 occurrences of the 5’CG3’ motif (the target for CpG Methyltransferase) on one strand. (B) Ava1 digests of DNA incubated
    Figure Legend Snippet: The 2905 bp DNA template (A) showing Ava1 restriction sites. The sequence contains 951 cytosines (32.2% C content) and 259 occurrences of the 5’CG3’ motif (the target for CpG Methyltransferase) on one strand. (B) Ava1 digests of DNA incubated

    Techniques Used: Sequencing, Incubation

    9) Product Images from "Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation"

    Article Title: Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029939

    Methylation of CpG sites in the vav 1 promoter impairs expression of the reporter gene in various cell lines. (A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p
    Figure Legend Snippet: Methylation of CpG sites in the vav 1 promoter impairs expression of the reporter gene in various cell lines. (A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p

    Techniques Used: Methylation, Expressing, Plasmid Preparation, Incubation, Transfection, Luciferase, Activity Assay

    10) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2010.01124.x

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
    Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    11) Product Images from "Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer"

    Article Title: Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer

    Journal: Oncogenesis

    doi: 10.1038/s41389-019-0135-1

    Non-CpHs methylation can silence HIF-1α gene transcription. a Analyzing in vitro DNA-methyltransferase, MSP I and MSss I recognition sites within HIF-1α gene promoter. b The methylation at CpC and CpG sites could cause transcriptional repression of HIF-1α gene by using luciferase activity assay. Data are presented as the means ± SD of three independent experiments. * p
    Figure Legend Snippet: Non-CpHs methylation can silence HIF-1α gene transcription. a Analyzing in vitro DNA-methyltransferase, MSP I and MSss I recognition sites within HIF-1α gene promoter. b The methylation at CpC and CpG sites could cause transcriptional repression of HIF-1α gene by using luciferase activity assay. Data are presented as the means ± SD of three independent experiments. * p

    Techniques Used: Methylation, In Vitro, Luciferase, Activity Assay

    12) Product Images from "Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat"

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    Journal: Journal of Neuroendocrinology

    doi: 10.1111/jne.12371

    Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro . The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in ( a ). The mutated plasmids were subsequently methylated by methyltransferase enzyme. ( b ) Successful methylation was determined using methylation sensitive restriction enzyme Pml I. (c, d ) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with ( c ) unmethylated and ( d ) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P
    Figure Legend Snippet: Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro . The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in ( a ). The mutated plasmids were subsequently methylated by methyltransferase enzyme. ( b ) Successful methylation was determined using methylation sensitive restriction enzyme Pml I. (c, d ) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with ( c ) unmethylated and ( d ) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P

    Techniques Used: Methylation, In Vitro, Overlap Extension Polymerase Chain Reaction, Mutagenesis, Luciferase, Cotransfection, Plasmid Preparation, Expressing

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P
    Figure Legend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Techniques Used: Methylation, Clone Assay

    Related Articles

    Methylation Sequencing:

    Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer
    Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using CpG methyltransferase Sss I (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment.

    Clone Assay:

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

    Amplification:

    Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum
    Article Snippet: Paragraph title: Amplicon-specific TAB pyrosequencing ... Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with CpG Methyltransferase (New England Biolabs, Ipswich, MA, USA) for a methylated control template.

    Article Title: Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3
    Article Snippet: SiHa cellular DNAs with and without a treatment by CpG methyltransferase SssI (New England Biolabs, Ipswich, MA) were included in each run of the assay as the methylated and unmethylated controls, respectively, to monitor the completeness of bisulfite conversion. .. SiHa cellular DNAs with and without a treatment by CpG methyltransferase SssI (New England Biolabs, Ipswich, MA) were included in each run of the assay as the methylated and unmethylated controls, respectively, to monitor the completeness of bisulfite conversion.

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: The concentration of converted DNA was adjusted to 5 ng μl−1 and DNA was amplified with the PyroMark PCR Kit (Qiagen) using a three-primer approach with a universal biotinylated primer . .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer
    Article Snippet: DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays
    Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the SssI (CpG) methylase (New England Biolabs, Beverly, MA), respectively, were used as U (unmethylated) and M (fully methylated) control DNA.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. Methylation specific PCR was carried out using promoter 1A of APC in two-step amplification procedure.

    Reporter Assay:

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: Paragraph title: In vitro methylated reporter assay ... Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

    Positive Control:

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control. .. Water blank was used as a negative control.

    Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer
    Article Snippet: After PCR amplification, products were analyzed by SSCP using the ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA) as described previously [ ], with Sss I methylase-treated normal DNA used as a positive control for CpG island methylation. .. Normal DNA samples were treated using CpG methyltransferase Sss I (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment.

    Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients
    Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with SssI methyltransferase (New England Biolabs: Ozyme, St-Quentin-Yvelines, France) and modified by bisulfite treatment. .. All raw and normalized data files for the microarray analysis have been deposited under accessions E-TABM-897 and E-TABM-898 at the European Bioinformatics Institute http://www.ebi.ac.uk/microarray-as/ae .

    Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines
    Article Snippet: In cases with diverging results from the two rounds of MSP, we did a third independent MSP round. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles. .. Water was used as a negative PCR control in both reactions.

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Human genomic DNA from peripheral blood lymphocytes, treated with bisulfite, was used as the negative control. .. Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control. .. The methylation status of the promoters was detected with MSP.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. Methylation specific PCR was carried out using promoter 1A of APC in two-step amplification procedure.

    Synthesized:

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

    Electrophoresis:

    Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer
    Article Snippet: DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles.

    Incubation:

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: The PCR condition consisted of one incubation of 10 minutes at 95°C, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 minutes. .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control.

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: The end-labeled probe was incubated with HeLa nuclear extract (5 µg; Millipore) for 40 minutes at room temperature in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and poly dI-dC), and resolved on a 5% native polyacrylamide gel. .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs).

    Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA
    Article Snippet: Sat2R: 5′–ATCAG ATTCC ATTCG AATCC ATTCG AAAAT GATTA CATTC GAATC CATTC GAAGA TTCCA TTTGA GCCTG TTCGA AAATT CCATT TGAGT CCAAC CAATG ATTCC ATTCA TTTCC ATTCA ATGAT TCCAT TCGAA TCCAT TTGGA T–3′. .. CpG methylation was introduced by an incubation with the bacterial DNA methyltransferase M.Sss I (New England BioLabs), in the presence of 160 µM S -adenosylmethionine (2 units µg−1 DNA) at 37°C for 16 h. The reaction was terminated by an incubation at 65°C for 30 min. .. The unmethylated satellite 2 DNA was cleaved with BstB I (10 units µg−1 DNA) at 65°C for 4 h, and the resulting methylated Sat2 DNA was purified by chromatography on TSKgel DEAE-5PW.

    Luciferase:

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Paragraph title: In vitro methylation, transient transfection and Luciferase assay ... M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Paragraph title: In vitro methylation, transient transfection and luciferase assay ... M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

    Activity Assay:

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

    Mass Spectrometry:

    Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer
    Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using CpG methyltransferase Sss I (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment.

    Modification:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul).

    Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum
    Article Snippet: The distribution of 5-hmC and 5-mC in the GAD1 promoter amplicon was determined by a modification of TAB-seq. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with CpG Methyltransferase (New England Biolabs, Ipswich, MA, USA) for a methylated control template.

    Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer
    Article Snippet: DNA samples were bisulphite treated using the MethylEasy Xceed Rapid DNA Bisulphite Modification kit (Human Genetic Signatures, Sydney, Australia) following the manufacturer's instructions. .. Normal DNA samples were treated using CpG methyltransferase Sss I (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment.

    Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer
    Article Snippet: Water blanks were included in each assay. .. DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. The reaction was performed in a total volume of 50 μl containing 10 μg of genomic DNA, 10 U of SssI methylase, 160 mM of S-adenosyl-metionina, 50 mM of NaCl, 10 mM of Tris-HCl, 10 mM of MgCl2 , 1 mM of DTT pH 7.9, during 18 hours at 37°C.

    Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients
    Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with SssI methyltransferase (New England Biolabs: Ozyme, St-Quentin-Yvelines, France) and modified by bisulfite treatment. .. All raw and normalized data files for the microarray analysis have been deposited under accessions E-TABM-897 and E-TABM-898 at the European Bioinformatics Institute http://www.ebi.ac.uk/microarray-as/ae .

    Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity
    Article Snippet: 0.5 µg of each complete plasmid were methylated via M.SssI methyltransferase (New England BioLabs, USA) or mock methylated (methyltransferase omitted). .. Each methylation reaction contained 640 mM S-adenosylmethionine and 16U M.SssI, and was incubated overnight at 37 °C and heat inactivated at 65 °C for 20 min.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. Methylation specific PCR was carried out using promoter 1A of APC in two-step amplification procedure.

    Whole Genome Amplification:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: For each assay, a standard curve of 0, 25, 50, 75 and 100% methylated DNA was measured. .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA. .. Primer sequences are listed in .

    Bisulfite Sequencing:

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Methylation analysis of CTCFL/BORIS NCBI RefSeq NR_072975, spanning nucleotides chr20:56099842-56100217 on GRCh37/hg19 assembly was performed in bisulphite sequencing assay. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Transfection:

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Paragraph title: In vitro methylation, transient transfection and Luciferase assay ... M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Paragraph title: In vitro methylation, transient transfection and luciferase assay ... M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Next, HEK293T cells were transiently transfected with unmethylated or methylated reporter plasmids by using the jetPEI reagent (Polyplus-transfection, New York, NY, USA).

    SYBR Green Assay:

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control.

    Generated:

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs). .. 200 ng of genomic DNA was treated using the EZ DNA Methylation-Direct Kit (Zymo Research) to convert unmethylated cytosines to uracil.

    CpG Methylation Assay:

    Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA
    Article Snippet: Sat2R: 5′–ATCAG ATTCC ATTCG AATCC ATTCG AAAAT GATTA CATTC GAATC CATTC GAAGA TTCCA TTTGA GCCTG TTCGA AAATT CCATT TGAGT CCAAC CAATG ATTCC ATTCA TTTCC ATTCA ATGAT TCCAT TCGAA TCCAT TTGGA T–3′. .. CpG methylation was introduced by an incubation with the bacterial DNA methyltransferase M.Sss I (New England BioLabs), in the presence of 160 µM S -adenosylmethionine (2 units µg−1 DNA) at 37°C for 16 h. The reaction was terminated by an incubation at 65°C for 30 min. .. The unmethylated satellite 2 DNA was cleaved with BstB I (10 units µg−1 DNA) at 65°C for 4 h, and the resulting methylated Sat2 DNA was purified by chromatography on TSKgel DEAE-5PW.

    Sequencing:

    Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum
    Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with CpG Methyltransferase (New England Biolabs, Ipswich, MA, USA) for a methylated control template.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Paragraph title: DNA extraction, sequencing and methylation analysis ... Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Binding Assay:

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: The end-labeled probe was incubated with HeLa nuclear extract (5 µg; Millipore) for 40 minutes at room temperature in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and poly dI-dC), and resolved on a 5% native polyacrylamide gel. .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs).

    Cellular Antioxidant Activity Assay:

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: The primer sequences for the methylated form were 5′-TGC GAG CGT TTT TTT AAA TGC-3′ (sense) and 5′-TAA ACT CGC AAA CCA ACT CG-3′ (antisense) (74 bp), and the primer sequences for the unmethylated form were 5′- GTT TGG TTG TTG TTT ATT GGT TG-3′ (sense) and 5′-CCT AAA CTC ACA AAC CAA CTC A-3′ (antisense) (115 bp). .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control.

    Magnetic Cell Separation:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    DNA Extraction:

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Paragraph title: DNA extraction, sequencing and methylation analysis ... Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Nucleic Acid Electrophoresis:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Fluorescence:

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The bisulfite-modified DNA was used as a template for fluorescence-based real-time PCR, as previously described [ ]. .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control.

    Methylation:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum
    Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with CpG Methyltransferase (New England Biolabs, Ipswich, MA, USA) for a methylated control template. .. To generate a 5-hmC-containing control, a 275-bp segment corresponding to 1635–1810 bp of pGEM1 (Promega) was amplified using linearized pGEM1 and 5-hydroxymethyl-2'- deoxycytidine-5'-triphosphate (Bioline,Taunton, MA, USA ) during PCR amplication., DNA from three ASD and three CONs were each immunoprecipitated (three IPs/patient) with either 5-hmC or 5-mC antibodies (as described above).

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control. .. Water blank was used as a negative control.

    Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer
    Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using CpG methyltransferase Sss I (New England Biolabs, Ipswich, MA), which methylates all CpGs before bisulfite treatment.

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs). .. 200 ng of genomic DNA was treated using the EZ DNA Methylation-Direct Kit (Zymo Research) to convert unmethylated cytosines to uracil.

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Next, HEK293T cells were transiently transfected with unmethylated or methylated reporter plasmids by using the jetPEI reagent (Polyplus-transfection, New York, NY, USA).

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: For each assay, a standard curve of 0, 25, 50, 75 and 100% methylated DNA was measured. .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA. .. Primer sequences are listed in .

    Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients
    Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with SssI methyltransferase (New England Biolabs: Ozyme, St-Quentin-Yvelines, France) and modified by bisulfite treatment. .. All raw and normalized data files for the microarray analysis have been deposited under accessions E-TABM-897 and E-TABM-898 at the European Bioinformatics Institute http://www.ebi.ac.uk/microarray-as/ae .

    Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines
    Article Snippet: In cases with diverging results from the two rounds of MSP, we did a third independent MSP round. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles. .. Water was used as a negative PCR control in both reactions.

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Paragraph title: Methylation-specific polymerase chain reaction (MSP) ... Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays
    Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the SssI (CpG) methylase (New England Biolabs, Beverly, MA), respectively, were used as U (unmethylated) and M (fully methylated) control DNA. .. Copy number of specific genes in samples was obtained using the standard curve generated from known amount of bisulfite-converted M sequence.

    Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity
    Article Snippet: Plasmid DNA from transformed liquid cultures was extracted using the QIAprep Spin Miniprep Kit (Qiagen, USA), and the direction of each insert within the plasmid was determined by Sanger sequencing using the forward primer previously listed for each region. .. 0.5 µg of each complete plasmid were methylated via M.SssI methyltransferase (New England BioLabs, USA) or mock methylated (methyltransferase omitted). .. Each methylation reaction contained 640 mM S-adenosylmethionine and 16U M.SssI, and was incubated overnight at 37 °C and heat inactivated at 65 °C for 20 min.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. Methylation specific PCR was carried out using promoter 1A of APC in two-step amplification procedure.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours. .. Methylation indexes (MI) were calculated as a percentage of the methylated CpGs compared to the total CpGs in each analysis.

    Mutagenesis:

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs).

    Isolation:

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Subcloning:

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: 200 ng of gDNA was treated in DNA methylation Direct kit by Zymo Research, following PCR by specific primers (available upon request) employing HotSTAR polymerase (Qiagen), and subcloning in pGEM-T-easy vector (Promega). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Size-exclusion Chromatography:

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Electrophoretic Mobility Shift Assay:

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay ... Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs).

    Purification:

    Article Title: Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3
    Article Snippet: DNA was extracted and purified from an aliquot of 200 µl of cervical swab sample in Specimen Transport Medium with QIAamp DNA Mini kit (Qiagen, Valentia, CA) and suspended in 100 µl AE buffer (10 mM Tris·Cl, 0.5 mM EDTA, pH 9.0). .. SiHa cellular DNAs with and without a treatment by CpG methyltransferase SssI (New England Biolabs, Ipswich, MA) were included in each run of the assay as the methylated and unmethylated controls, respectively, to monitor the completeness of bisulfite conversion.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Next, HEK293T cells were transiently transfected with unmethylated or methylated reporter plasmids by using the jetPEI reagent (Polyplus-transfection, New York, NY, USA).

    Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA
    Article Snippet: The DNA fragments were purified by the same methods as described above. .. CpG methylation was introduced by an incubation with the bacterial DNA methyltransferase M.Sss I (New England BioLabs), in the presence of 160 µM S -adenosylmethionine (2 units µg−1 DNA) at 37°C for 16 h. The reaction was terminated by an incubation at 65°C for 30 min.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours. .. Methylation indexes (MI) were calculated as a percentage of the methylated CpGs compared to the total CpGs in each analysis.

    Polymerase Chain Reaction:

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control.

    Article Title: Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3
    Article Snippet: SiHa cellular DNAs with and without a treatment by CpG methyltransferase SssI (New England Biolabs, Ipswich, MA) were included in each run of the assay as the methylated and unmethylated controls, respectively, to monitor the completeness of bisulfite conversion. .. SiHa cellular DNAs with and without a treatment by CpG methyltransferase SssI (New England Biolabs, Ipswich, MA) were included in each run of the assay as the methylated and unmethylated controls, respectively, to monitor the completeness of bisulfite conversion.

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: A 231-bp PCR fragment (position −222 to +9 relative to the initiation codon) containing the putative CTCF binding site in the 5′UTR of the FXN gene was used as the probe. .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs).

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer
    Article Snippet: Paragraph title: Methylation-Specific Polymerase Chain Reaction (MSP) ... DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles.

    Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients
    Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with SssI methyltransferase (New England Biolabs: Ozyme, St-Quentin-Yvelines, France) and modified by bisulfite treatment.

    Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines
    Article Snippet: Paragraph title: Methylation-specific PCR (MSP) ... Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles.

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Paragraph title: Methylation-specific polymerase chain reaction (MSP) ... Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays
    Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the SssI (CpG) methylase (New England Biolabs, Beverly, MA), respectively, were used as U (unmethylated) and M (fully methylated) control DNA.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: Paragraph title: Methylation Specific PCR ... In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours. .. Methylation indexes (MI) were calculated as a percentage of the methylated CpGs compared to the total CpGs in each analysis.

    FACS:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Activated Clotting Time Assay:

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: The primer sequences for the methylated form were 5′-TGC GAG CGT TTT TTT AAA TGC-3′ (sense) and 5′-TAA ACT CGC AAA CCA ACT CG-3′ (antisense) (74 bp), and the primer sequences for the unmethylated form were 5′- GTT TGG TTG TTG TTT ATT GGT TG-3′ (sense) and 5′-CCT AAA CTC ACA AAC CAA CTC A-3′ (antisense) (115 bp). .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control.

    Hot Start PCR:

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control. .. The primers for the methylated and unmethylated DNA sequences are listed in Table .

    Plasmid Preparation:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylation was confirmed by digestion with the methylation-sensitive restriction enzymes Hha I and Hpa II.

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylation was confirmed by digestion with the methylation-sensitive restriction enzymes HhaI and HpaII.

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Next, HEK293T cells were transiently transfected with unmethylated or methylated reporter plasmids by using the jetPEI reagent (Polyplus-transfection, New York, NY, USA).

    Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA
    Article Snippet: For the Sat2L and Sat2R DNA fragments, eight Sat2L (145 base pairs) or Sat2R (146 base pairs) DNA fragments were tandemly ligated into the pGEM-T Easy vector. .. CpG methylation was introduced by an incubation with the bacterial DNA methyltransferase M.Sss I (New England BioLabs), in the presence of 160 µM S -adenosylmethionine (2 units µg−1 DNA) at 37°C for 16 h. The reaction was terminated by an incubation at 65°C for 30 min.

    Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity
    Article Snippet: Plasmid DNA from transformed liquid cultures was extracted using the QIAprep Spin Miniprep Kit (Qiagen, USA), and the direction of each insert within the plasmid was determined by Sanger sequencing using the forward primer previously listed for each region. .. 0.5 µg of each complete plasmid were methylated via M.SssI methyltransferase (New England BioLabs, USA) or mock methylated (methyltransferase omitted). .. Each methylation reaction contained 640 mM S-adenosylmethionine and 16U M.SssI, and was incubated overnight at 37 °C and heat inactivated at 65 °C for 20 min.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: 200 ng of gDNA was treated in DNA methylation Direct kit by Zymo Research, following PCR by specific primers (available upon request) employing HotSTAR polymerase (Qiagen), and subcloning in pGEM-T-easy vector (Promega). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Software:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Sequences were inspected with Sequence Scanner software 1.0 (Applied Biosystems), excluding variants present in the SNP database (dbSNP) http://www.ncbi.nlm.nih.gov/snp ). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Real-time Polymerase Chain Reaction:

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The bisulfite-modified DNA was used as a template for fluorescence-based real-time PCR, as previously described [ ]. .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control.

    Negative Control:

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Human genomic DNA from peripheral blood lymphocytes, treated with bisulfite, was used as the negative control. .. Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control.

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker
    Article Snippet: In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency.

    Recombinant:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: Paragraph title: Methylation of the Recombinant Plasmids ... The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul).

    In Vitro:

    Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum
    Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with CpG Methyltransferase (New England Biolabs, Ipswich, MA, USA) for a methylated control template. .. To generate a 5-hmC-containing control, a 275-bp segment corresponding to 1635–1810 bp of pGEM1 (Promega) was amplified using linearized pGEM1 and 5-hydroxymethyl-2'- deoxycytidine-5'-triphosphate (Bioline,Taunton, MA, USA ) during PCR amplication., DNA from three ASD and three CONs were each immunoprecipitated (three IPs/patient) with either 5-hmC or 5-mC antibodies (as described above).

    Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma
    Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by CpG methyltransferase (Sss I) following the manufacturer's directions (New England BioLabs, Inc., Beverly, MA), was used as a positive control. .. Water blank was used as a negative control.

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
    Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using M.SssI CpG methyltransferase and S-adenosylmethionine (New England BioLabs). .. 200 ng of genomic DNA was treated using the EZ DNA Methylation-Direct Kit (Zymo Research) to convert unmethylated cytosines to uracil.

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients
    Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ). .. Next, HEK293T cells were transiently transfected with unmethylated or methylated reporter plasmids by using the jetPEI reagent (Polyplus-transfection, New York, NY, USA).

    Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines
    Article Snippet: In cases with diverging results from the two rounds of MSP, we did a third independent MSP round. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles. .. Water was used as a negative PCR control in both reactions.

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: Human genomic DNA from peripheral blood lymphocytes, treated with bisulfite, was used as the negative control. .. Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control. .. The methylation status of the promoters was detected with MSP.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control. .. Each plate included subject DNA samples, water blanks, and serial dilutions (30–0.003 ng) of the positive control, which were used to construct a calibration curve.

    Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays
    Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the SssI (CpG) methylase (New England Biolabs, Beverly, MA), respectively, were used as U (unmethylated) and M (fully methylated) control DNA. .. Copy number of specific genes in samples was obtained using the standard curve generated from known amount of bisulfite-converted M sequence.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours. .. Methylation indexes (MI) were calculated as a percentage of the methylated CpGs compared to the total CpGs in each analysis.

    DNA Methylation Assay:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach
    Article Snippet: The methylation status of the MLH1 , MGMT , and DAPK-1 promoters was determined by treating the DNA with bisulfite, using the EZ DNA Methylation Gold Kit™ (Zymo Research), according to the manufacturer’s instructions. .. Human genomic DNA treated in vitro with SssI methyltransferase (New England Biolabs, UK) was used as the positive control.

    Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene
    Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the SssI CpG methylase (New England Biolabs, United Kingdom) at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with additional SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours.

    Concentration Assay:

    Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease
    Article Snippet: The concentration of converted DNA was adjusted to 5 ng μl−1 and DNA was amplified with the PyroMark PCR Kit (Qiagen) using a three-primer approach with a universal biotinylated primer . .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA.

    Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer
    Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Beverly, MA) to generate completely methylated DNA as a positive control.

    Staining:

    Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer
    Article Snippet: DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles.

    Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients
    Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with SssI methyltransferase (New England Biolabs: Ozyme, St-Quentin-Yvelines, France) and modified by bisulfite treatment.

    Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines
    Article Snippet: PCR products were loaded onto 7.5% polyacrylamide gels, stained with ethidium bromide, and visualized by UV illumination. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles.

    Variant Assay:

    Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells
    Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of M.SssI CpG methyltransferase (New England BioLabs, Ipswich, MA, USA) in vitro at 37°C for 20 h. After purification with the DNA Clean & Concentrator™ -5 Kit (Zymo Research) according to the manufacturer’s protocol, methylation of the plasmid was verified by bisulfite pyrosequencing of the selected CpG units in the DEFB1 promoter ( ).

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  • 99
    New England Biolabs m sss
    CpG methylation of nucleosomal <t>DNA.</t> (A) Scheme for the preparation of CpG‐methylated nucleosomal DNA. The 146‐bp human α‐satellite nucleosomal DNA 18 was mutated to contain four CpG dinucleotide‐containing CACGTG sequences, which are recognized by the methylation‐sensitive restriction enzyme Eco72 I. The DNA designated as CpG146 is biochemically methylated by <t>M.Sss</t> I, and then digested by Eco72 I, which is a CpG methylation‐sensitive restriction endonuclease for the CACGTG sequence. The full‐length 146‐bp nucleosomal DNA is methylated by M.Sss I, and a portion of the methylated DNA is examined by a digestion with Eco 72I before the NCP reconstitution. (B) Sequences of nucleosomal DNAs used for crystal structure analyses. NCP146, 146‐bp α‐satellite nucleosomal DNA 18 ; and CpG146, CpG dinucleotide sequence‐introduced 146‐bp nucleosomal DNA (this study). Four CpG dinucleotide‐containing Eco72 I recognition sequences, created by the site‐directed mutagenesis of NCP146, are shown in green. The positions of the CpG dinucleotides are underlined. Sat2R: the 145‐bp satellite 2 derivative right nucleosomal DNA 17 . Sat2L: the 146‐bp satellite 2 derivative left nucleosomal DNA 17 . The relative positions of DNA bases from the dyad axis (0) are indicated from −70 to +70 at the top. Minor groove‐inward facing regions, as reported by Chua et al . 31 , are boxed within blue squares. The major grooves of the boxed DNA sequences are outward‐facing, and CpG‐methyl reader and/or eraser proteins can access 5mC. (C) Eco72 I digestion patterns of double‐stranded CpG‐containing oligonucleotides. Oligonucleotides (a) and (b), or (c) and (d), which are both derived from the CpG146 sequence, were annealed to each other in the presence or absence of 5mC, at the indicated positions in bold letters. Lane 1, nondigested dsDNA; lanes 2–5, dsDNA digested with 1.8 units·pmol −1 Eco72 I for 16 h. Lane 2, nonmethylated dsDNA; lanes 3 and 4, hemimethylated dsDNAs; and lane 5, fully methylated dsDNA. The superscript m at the top indicates a 5mC‐containing oligonucleotide. The DNA bands at 21‐bp and 10‐bp in the left panel and the DNA band at 18‐bp in the right panel indicate Eco72 I‐digested DNA fragments.
    M Sss, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs gpc mtase buffer
    Heatmaps of average <t>GpC</t> and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates <t>MTase</t> treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009
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    New England Biolabs cpg
    Representative results of the quantitations of the methylation levels by Multiplex-BSP-seq for health control (HC), chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) plasma <t>DNA</t> samples. For each gene, the heat map of the methylation patterns for each <t>CpG</t> is shown. The methylation level (%) measured at each individual CpG site is expressed by the percentage of methylated CpG versus unmethylated CpG sites. HC, CHB, LC and HCC are represented by colored areas of blue, green, violet and red, respectively. The colored area is defined by 25%/75% quantiles. The comparison of the CpG methylation levels for each stage is colored blue to red, in small squares, to indicate the different P values. Representative heat map and methylation plot analysis of 5 target genes; consistently low levels of methylation of all CpGs were observed in GAPDH and steady high levels of CpG(CG1,2 and 7) methylation in KCNV1 are independent of the HCC developmental stage, while the methylation statuses of the other 3 genes (ZNF300, SLC22A20 and SHISA7) varied according to the developmental stage.
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    CpG methylation of nucleosomal DNA. (A) Scheme for the preparation of CpG‐methylated nucleosomal DNA. The 146‐bp human α‐satellite nucleosomal DNA 18 was mutated to contain four CpG dinucleotide‐containing CACGTG sequences, which are recognized by the methylation‐sensitive restriction enzyme Eco72 I. The DNA designated as CpG146 is biochemically methylated by M.Sss I, and then digested by Eco72 I, which is a CpG methylation‐sensitive restriction endonuclease for the CACGTG sequence. The full‐length 146‐bp nucleosomal DNA is methylated by M.Sss I, and a portion of the methylated DNA is examined by a digestion with Eco 72I before the NCP reconstitution. (B) Sequences of nucleosomal DNAs used for crystal structure analyses. NCP146, 146‐bp α‐satellite nucleosomal DNA 18 ; and CpG146, CpG dinucleotide sequence‐introduced 146‐bp nucleosomal DNA (this study). Four CpG dinucleotide‐containing Eco72 I recognition sequences, created by the site‐directed mutagenesis of NCP146, are shown in green. The positions of the CpG dinucleotides are underlined. Sat2R: the 145‐bp satellite 2 derivative right nucleosomal DNA 17 . Sat2L: the 146‐bp satellite 2 derivative left nucleosomal DNA 17 . The relative positions of DNA bases from the dyad axis (0) are indicated from −70 to +70 at the top. Minor groove‐inward facing regions, as reported by Chua et al . 31 , are boxed within blue squares. The major grooves of the boxed DNA sequences are outward‐facing, and CpG‐methyl reader and/or eraser proteins can access 5mC. (C) Eco72 I digestion patterns of double‐stranded CpG‐containing oligonucleotides. Oligonucleotides (a) and (b), or (c) and (d), which are both derived from the CpG146 sequence, were annealed to each other in the presence or absence of 5mC, at the indicated positions in bold letters. Lane 1, nondigested dsDNA; lanes 2–5, dsDNA digested with 1.8 units·pmol −1 Eco72 I for 16 h. Lane 2, nonmethylated dsDNA; lanes 3 and 4, hemimethylated dsDNAs; and lane 5, fully methylated dsDNA. The superscript m at the top indicates a 5mC‐containing oligonucleotide. The DNA bands at 21‐bp and 10‐bp in the left panel and the DNA band at 18‐bp in the right panel indicate Eco72 I‐digested DNA fragments.

    Journal: FEBS Open Bio

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

    doi: 10.1002/2211-5463.12064

    Figure Lengend Snippet: CpG methylation of nucleosomal DNA. (A) Scheme for the preparation of CpG‐methylated nucleosomal DNA. The 146‐bp human α‐satellite nucleosomal DNA 18 was mutated to contain four CpG dinucleotide‐containing CACGTG sequences, which are recognized by the methylation‐sensitive restriction enzyme Eco72 I. The DNA designated as CpG146 is biochemically methylated by M.Sss I, and then digested by Eco72 I, which is a CpG methylation‐sensitive restriction endonuclease for the CACGTG sequence. The full‐length 146‐bp nucleosomal DNA is methylated by M.Sss I, and a portion of the methylated DNA is examined by a digestion with Eco 72I before the NCP reconstitution. (B) Sequences of nucleosomal DNAs used for crystal structure analyses. NCP146, 146‐bp α‐satellite nucleosomal DNA 18 ; and CpG146, CpG dinucleotide sequence‐introduced 146‐bp nucleosomal DNA (this study). Four CpG dinucleotide‐containing Eco72 I recognition sequences, created by the site‐directed mutagenesis of NCP146, are shown in green. The positions of the CpG dinucleotides are underlined. Sat2R: the 145‐bp satellite 2 derivative right nucleosomal DNA 17 . Sat2L: the 146‐bp satellite 2 derivative left nucleosomal DNA 17 . The relative positions of DNA bases from the dyad axis (0) are indicated from −70 to +70 at the top. Minor groove‐inward facing regions, as reported by Chua et al . 31 , are boxed within blue squares. The major grooves of the boxed DNA sequences are outward‐facing, and CpG‐methyl reader and/or eraser proteins can access 5mC. (C) Eco72 I digestion patterns of double‐stranded CpG‐containing oligonucleotides. Oligonucleotides (a) and (b), or (c) and (d), which are both derived from the CpG146 sequence, were annealed to each other in the presence or absence of 5mC, at the indicated positions in bold letters. Lane 1, nondigested dsDNA; lanes 2–5, dsDNA digested with 1.8 units·pmol −1 Eco72 I for 16 h. Lane 2, nonmethylated dsDNA; lanes 3 and 4, hemimethylated dsDNAs; and lane 5, fully methylated dsDNA. The superscript m at the top indicates a 5mC‐containing oligonucleotide. The DNA bands at 21‐bp and 10‐bp in the left panel and the DNA band at 18‐bp in the right panel indicate Eco72 I‐digested DNA fragments.

    Article Snippet: The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments.

    Techniques: CpG Methylation Assay, Methylation, Sequencing, Mutagenesis, Derivative Assay

    Binding analysis between MBD2 and nucleosomal DNAs. (A) Scheme of the binding analysis. (B–D) Results of the MBD2‐binding analysis. The nucleosomal DNAs used in the assay are as follows: (B) M.Sss I‐treated146‐bp α‐satellite DNA (NCP146); (C) M.Sss I‐untreated 146‐bp α‐satellite‐based DNA containing four CpG sites (CpG146); and (D) M.Sss I‐treated CpG146 DNA. Lane 1, Input nucleosomal DNA (125 ng); lane 2, flow‐through fraction after the incubation of nucleosomal DNA with immobilized MBD2; lane 3, wash fraction of the first washing step; lane 4, wash fraction of the fourth washing step; lane 5, eluted fraction. In each panel, the position of the 100‐bp DNA is indicated on the right, and the position of the nucleosomal DNA is indicated by a red arrow.

    Journal: FEBS Open Bio

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

    doi: 10.1002/2211-5463.12064

    Figure Lengend Snippet: Binding analysis between MBD2 and nucleosomal DNAs. (A) Scheme of the binding analysis. (B–D) Results of the MBD2‐binding analysis. The nucleosomal DNAs used in the assay are as follows: (B) M.Sss I‐treated146‐bp α‐satellite DNA (NCP146); (C) M.Sss I‐untreated 146‐bp α‐satellite‐based DNA containing four CpG sites (CpG146); and (D) M.Sss I‐treated CpG146 DNA. Lane 1, Input nucleosomal DNA (125 ng); lane 2, flow‐through fraction after the incubation of nucleosomal DNA with immobilized MBD2; lane 3, wash fraction of the first washing step; lane 4, wash fraction of the fourth washing step; lane 5, eluted fraction. In each panel, the position of the 100‐bp DNA is indicated on the right, and the position of the nucleosomal DNA is indicated by a red arrow.

    Article Snippet: The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments.

    Techniques: Binding Assay, Flow Cytometry, Incubation

    Biochemical methylation of CpG dinucleotide‐containing nucleosomal DNA (CpG146). Lanes are as follows: M, 10‐bp DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA; cat. 10821‐015); (+), presence; and (−), absence of the respective enzyme shown on the left. (A) Comparison of the CpG methyltransferase M.Sss I enzymatic activities. CpG146 DNA was methylated with M.Sss I enzymes as follows: lanes 3–5, M.Sss I purchased from New England Biolabs (NEB, cat. M0226M); and lanes 6–11, the M.Sss I protein purified in this study. In lanes 1 and 2, CpG146 DNAs were incubated in the methylation reaction buffer, in the absence of M.Sss I. Each DNA sample was methylated with an increasing amount of M.Sss I, and the DNA from each reaction was purified, digested with the methyl‐sensitive restriction endonuclease Eco72 I, and electrophoresed. The specific units of the NEB M.Sss I enzyme used in each reaction are as follows: lane 3, 2.0 units·μg −1 DNA; lane 4, 3.0 units·μg −1 DNA; and lane 5, 4.0 units·μg −1 DNA. The amount of the purified M.Sss I protein used in each reaction is as follows: lane 6, 0.032 μg·μg −1 DNA; lane 7, 0.063 μg·μg −1 DNA; lane 8, 0.13 μg·μg −1 DNA; lane 9, 0.25 μg·μg −1 DNA; lane 10, 0.50 μg·μg −1 DNA; and lane 11, 1.0 μg·μg −1 DNA. The positions of the 146‐bp CpG146 nucleosomal DNA, the half unit of CpG146, and the digested DNAs are shown by a red arrow, a black arrow, and a gray square bracket, respectively. (B) Confirmation of the CpG‐methylated CpG146 DNA by Eco72 I digestion. The digestion was performed with 20 units·μg −1 DNA (1.8 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐sensitive restriction enzyme Eco72 I. (C) Confirmation of the CpG‐methylated CpG146 DNA by Msp JI digestion. The digestion was performed with 5 units·μg −1 DNA (0.45 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐dependent restriction enzyme Msp JI.

    Journal: FEBS Open Bio

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

    doi: 10.1002/2211-5463.12064

    Figure Lengend Snippet: Biochemical methylation of CpG dinucleotide‐containing nucleosomal DNA (CpG146). Lanes are as follows: M, 10‐bp DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA; cat. 10821‐015); (+), presence; and (−), absence of the respective enzyme shown on the left. (A) Comparison of the CpG methyltransferase M.Sss I enzymatic activities. CpG146 DNA was methylated with M.Sss I enzymes as follows: lanes 3–5, M.Sss I purchased from New England Biolabs (NEB, cat. M0226M); and lanes 6–11, the M.Sss I protein purified in this study. In lanes 1 and 2, CpG146 DNAs were incubated in the methylation reaction buffer, in the absence of M.Sss I. Each DNA sample was methylated with an increasing amount of M.Sss I, and the DNA from each reaction was purified, digested with the methyl‐sensitive restriction endonuclease Eco72 I, and electrophoresed. The specific units of the NEB M.Sss I enzyme used in each reaction are as follows: lane 3, 2.0 units·μg −1 DNA; lane 4, 3.0 units·μg −1 DNA; and lane 5, 4.0 units·μg −1 DNA. The amount of the purified M.Sss I protein used in each reaction is as follows: lane 6, 0.032 μg·μg −1 DNA; lane 7, 0.063 μg·μg −1 DNA; lane 8, 0.13 μg·μg −1 DNA; lane 9, 0.25 μg·μg −1 DNA; lane 10, 0.50 μg·μg −1 DNA; and lane 11, 1.0 μg·μg −1 DNA. The positions of the 146‐bp CpG146 nucleosomal DNA, the half unit of CpG146, and the digested DNAs are shown by a red arrow, a black arrow, and a gray square bracket, respectively. (B) Confirmation of the CpG‐methylated CpG146 DNA by Eco72 I digestion. The digestion was performed with 20 units·μg −1 DNA (1.8 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐sensitive restriction enzyme Eco72 I. (C) Confirmation of the CpG‐methylated CpG146 DNA by Msp JI digestion. The digestion was performed with 5 units·μg −1 DNA (0.45 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐dependent restriction enzyme Msp JI.

    Article Snippet: The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments.

    Techniques: Methylation, Purification, Incubation

    Native PAGE of the NCP samples used for crystallization. The positions of the NCPs and the free CpG146 DNAs are shown by red square brackets and a red arrow, respectively. (A) Ethidium bromide‐stained gel. (B) CBB‐stained gel. Lane M, 10‐bp DNA ladder (Thermo Fisher Scientific; cat. 10821‐015). Lanes 1–4, nucleosome core particles containing CpG146 DNA untreated with CpG methyltransferase M.Sss I. Lanes 5–8, nucleosome core particles containing CpG146 DNA treated with M.Sss I. Lanes 1 and 5, NCP samples after reconstitution and dialysis. Lanes 2 and 6, NCP samples after heat treatment. Lanes 3 and 7, supernatant fractions of MgCl 2 ‐treated NCP samples. Lanes 4 and 8, precipitated fractions of the MgCl 2 ‐treated NCP samples that were used for crystallization.

    Journal: FEBS Open Bio

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

    doi: 10.1002/2211-5463.12064

    Figure Lengend Snippet: Native PAGE of the NCP samples used for crystallization. The positions of the NCPs and the free CpG146 DNAs are shown by red square brackets and a red arrow, respectively. (A) Ethidium bromide‐stained gel. (B) CBB‐stained gel. Lane M, 10‐bp DNA ladder (Thermo Fisher Scientific; cat. 10821‐015). Lanes 1–4, nucleosome core particles containing CpG146 DNA untreated with CpG methyltransferase M.Sss I. Lanes 5–8, nucleosome core particles containing CpG146 DNA treated with M.Sss I. Lanes 1 and 5, NCP samples after reconstitution and dialysis. Lanes 2 and 6, NCP samples after heat treatment. Lanes 3 and 7, supernatant fractions of MgCl 2 ‐treated NCP samples. Lanes 4 and 8, precipitated fractions of the MgCl 2 ‐treated NCP samples that were used for crystallization.

    Article Snippet: The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments.

    Techniques: Clear Native PAGE, Crystallization Assay, Staining

    Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, CpG Methylation Assay, Methylation

    Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography

    Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, Methylation

    Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Journal: eLife

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    doi: 10.7554/eLife.23203

    Figure Lengend Snippet: Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Article Snippet: One million nuclei were resupended in reaction buffer to yield a suspension with a final concentration of 1x GpC MTase buffer (NEB), 0.32 mM S-Adenosylmethionine (SAM) (NEB), and 50 ul of GpC methyltransferase (4 U/ul)) from M.CviPI (NEB).

    Techniques: Gel Permeation Chromatography, Methylation, CpG Methylation Assay, Activity Assay

    Representative results of the quantitations of the methylation levels by Multiplex-BSP-seq for health control (HC), chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) plasma DNA samples. For each gene, the heat map of the methylation patterns for each CpG is shown. The methylation level (%) measured at each individual CpG site is expressed by the percentage of methylated CpG versus unmethylated CpG sites. HC, CHB, LC and HCC are represented by colored areas of blue, green, violet and red, respectively. The colored area is defined by 25%/75% quantiles. The comparison of the CpG methylation levels for each stage is colored blue to red, in small squares, to indicate the different P values. Representative heat map and methylation plot analysis of 5 target genes; consistently low levels of methylation of all CpGs were observed in GAPDH and steady high levels of CpG(CG1,2 and 7) methylation in KCNV1 are independent of the HCC developmental stage, while the methylation statuses of the other 3 genes (ZNF300, SLC22A20 and SHISA7) varied according to the developmental stage.

    Journal: Clinical Epigenetics

    Article Title: Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma

    doi: 10.1186/1868-7083-6-30

    Figure Lengend Snippet: Representative results of the quantitations of the methylation levels by Multiplex-BSP-seq for health control (HC), chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) plasma DNA samples. For each gene, the heat map of the methylation patterns for each CpG is shown. The methylation level (%) measured at each individual CpG site is expressed by the percentage of methylated CpG versus unmethylated CpG sites. HC, CHB, LC and HCC are represented by colored areas of blue, green, violet and red, respectively. The colored area is defined by 25%/75% quantiles. The comparison of the CpG methylation levels for each stage is colored blue to red, in small squares, to indicate the different P values. Representative heat map and methylation plot analysis of 5 target genes; consistently low levels of methylation of all CpGs were observed in GAPDH and steady high levels of CpG(CG1,2 and 7) methylation in KCNV1 are independent of the HCC developmental stage, while the methylation statuses of the other 3 genes (ZNF300, SLC22A20 and SHISA7) varied according to the developmental stage.

    Article Snippet: The in vitro methylated DNA from the HepG2 cells was obtained using the CpG (M. SssI) methyltransferase (NEB, MA,USA) treatment and used as a positive control.

    Techniques: Methylation, Multiplex Assay, Infection, Liquid Chromatography, CpG Methylation Assay

    Receiver operating characteristics (ROC) and multiple univariate logistic regression analyses for using CpGs to distinguish between hepatocellular carcinoma (HCC) developmental stages are shown according to the CpG position and disease stage (HC + CHB versus LC + HCC or HC + CHB + LC versus HCC). (A) Receiver operating characteristic (ROC) curves for ZNF300, SLC22A20 and SHISA7. Complete DNA methylation data from all four stages of HCC development were used to construct the ROC curves. The ROC curves plot the sensitivity versus 100-specificity. Upper panel: a lower cut-off value was used to distinguish between (LC + HCC)/ (HC + CHB).Lower panel: a higher cut-off value was used to distinguish between HCC/(HC + CHB + LC). (B) A multiple univariate logistic regression analysis was performed using the CpG methylation patterns to evaluate the association between gene methylation and the stage of HCC development. Relationship between the CpG methylation (odds ratios) and the developmental stage. To separate (LC + HCC)/ (HC + CHB) and HCC/(HC + CHB + LC), both univariate (which considers the methylation levels) and multivariate (which also considers the age and gender) logistic regressions were performed using CpG methylation data for ZNF300, SLC22A20 and SHISA7.

    Journal: Clinical Epigenetics

    Article Title: Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma

    doi: 10.1186/1868-7083-6-30

    Figure Lengend Snippet: Receiver operating characteristics (ROC) and multiple univariate logistic regression analyses for using CpGs to distinguish between hepatocellular carcinoma (HCC) developmental stages are shown according to the CpG position and disease stage (HC + CHB versus LC + HCC or HC + CHB + LC versus HCC). (A) Receiver operating characteristic (ROC) curves for ZNF300, SLC22A20 and SHISA7. Complete DNA methylation data from all four stages of HCC development were used to construct the ROC curves. The ROC curves plot the sensitivity versus 100-specificity. Upper panel: a lower cut-off value was used to distinguish between (LC + HCC)/ (HC + CHB).Lower panel: a higher cut-off value was used to distinguish between HCC/(HC + CHB + LC). (B) A multiple univariate logistic regression analysis was performed using the CpG methylation patterns to evaluate the association between gene methylation and the stage of HCC development. Relationship between the CpG methylation (odds ratios) and the developmental stage. To separate (LC + HCC)/ (HC + CHB) and HCC/(HC + CHB + LC), both univariate (which considers the methylation levels) and multivariate (which also considers the age and gender) logistic regressions were performed using CpG methylation data for ZNF300, SLC22A20 and SHISA7.

    Article Snippet: The in vitro methylated DNA from the HepG2 cells was obtained using the CpG (M. SssI) methyltransferase (NEB, MA,USA) treatment and used as a positive control.

    Techniques: Liquid Chromatography, DNA Methylation Assay, Construct, CpG Methylation Assay, Methylation