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    New England Biolabs escherichiacoli endonuclease viii
    AID can process blunt DSBs to generate staggered DSBs. A 59 bp DNA fragment containing 13 RGYW repeats was cloned into pCR-Blunt II-TOPO ® vector. A 191 bp 5'-phosphorylated blunt-ended linearized DNA substrate and the dU contained control DNA substrate were generated by PCR amplification of the pCR-Blunt IITOPO® vector containing the 59 bp fragment DNA using the forward 5'-phosphorylated primer A or primer A1 (containing a dU instead of a dC at position 3), specific for the 5' region of the 59 bp DNA fragment and the reverse primer B, specific for the vector sequence 116 bp downstream of the inserted 59 bp DNA fragment. The linearized DNA substrate was incubated with nil or recombinant GST-mouse AID fusion protein, then treated with SAP and Antarctic Phosphatase. These catalyze the release of 5'-phosphate groups from DNA yielding non-phosphorylated DNA ends, which cannot been amplified by LM-PCR. Incubation of DNA pretreated with nil or AID with recombinant E. coli Ung, followed by treatment with <t>Endo</t> <t>VIII</t> showed that AID could process blunt DSBs to yield staggered DSB ends. Such staggered DSB ends were amplified by specific LM-PCR after treatment with T4 pol.
    Escherichiacoli Endonuclease Viii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ape1 new england biolabs
    AID can process blunt DSBs to generate staggered DSBs. A 59 bp DNA fragment containing 13 RGYW repeats was cloned into pCR-Blunt II-TOPO ® vector. A 191 bp 5'-phosphorylated blunt-ended linearized DNA substrate and the dU contained control DNA substrate were generated by PCR amplification of the pCR-Blunt IITOPO® vector containing the 59 bp fragment DNA using the forward 5'-phosphorylated primer A or primer A1 (containing a dU instead of a dC at position 3), specific for the 5' region of the 59 bp DNA fragment and the reverse primer B, specific for the vector sequence 116 bp downstream of the inserted 59 bp DNA fragment. The linearized DNA substrate was incubated with nil or recombinant GST-mouse AID fusion protein, then treated with SAP and Antarctic Phosphatase. These catalyze the release of 5'-phosphate groups from DNA yielding non-phosphorylated DNA ends, which cannot been amplified by LM-PCR. Incubation of DNA pretreated with nil or AID with recombinant E. coli Ung, followed by treatment with <t>Endo</t> <t>VIII</t> showed that AID could process blunt DSBs to yield staggered DSB ends. Such staggered DSB ends were amplified by specific LM-PCR after treatment with T4 pol.
    Ape1 New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs endonuclease viii endoviii
    AID can process blunt DSBs to generate staggered DSBs. A 59 bp DNA fragment containing 13 RGYW repeats was cloned into pCR-Blunt II-TOPO ® vector. A 191 bp 5'-phosphorylated blunt-ended linearized DNA substrate and the dU contained control DNA substrate were generated by PCR amplification of the pCR-Blunt IITOPO® vector containing the 59 bp fragment DNA using the forward 5'-phosphorylated primer A or primer A1 (containing a dU instead of a dC at position 3), specific for the 5' region of the 59 bp DNA fragment and the reverse primer B, specific for the vector sequence 116 bp downstream of the inserted 59 bp DNA fragment. The linearized DNA substrate was incubated with nil or recombinant GST-mouse AID fusion protein, then treated with SAP and Antarctic Phosphatase. These catalyze the release of 5'-phosphate groups from DNA yielding non-phosphorylated DNA ends, which cannot been amplified by LM-PCR. Incubation of DNA pretreated with nil or AID with recombinant E. coli Ung, followed by treatment with <t>Endo</t> <t>VIII</t> showed that AID could process blunt DSBs to yield staggered DSB ends. Such staggered DSB ends were amplified by specific LM-PCR after treatment with T4 pol.
    Endonuclease Viii Endoviii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AID can process blunt DSBs to generate staggered DSBs. A 59 bp DNA fragment containing 13 RGYW repeats was cloned into pCR-Blunt II-TOPO ® vector. A 191 bp 5'-phosphorylated blunt-ended linearized DNA substrate and the dU contained control DNA substrate were generated by PCR amplification of the pCR-Blunt IITOPO® vector containing the 59 bp fragment DNA using the forward 5'-phosphorylated primer A or primer A1 (containing a dU instead of a dC at position 3), specific for the 5' region of the 59 bp DNA fragment and the reverse primer B, specific for the vector sequence 116 bp downstream of the inserted 59 bp DNA fragment. The linearized DNA substrate was incubated with nil or recombinant GST-mouse AID fusion protein, then treated with SAP and Antarctic Phosphatase. These catalyze the release of 5'-phosphate groups from DNA yielding non-phosphorylated DNA ends, which cannot been amplified by LM-PCR. Incubation of DNA pretreated with nil or AID with recombinant E. coli Ung, followed by treatment with Endo VIII showed that AID could process blunt DSBs to yield staggered DSB ends. Such staggered DSB ends were amplified by specific LM-PCR after treatment with T4 pol.

    Journal: Molecular immunology

    Article Title: AID- and Ung-dependent generation of staggered double-strand DNA breaks in immunoglobulin class switch DNA recombination: a post-cleavage role for AID

    doi: 10.1016/j.molimm.2008.07.003

    Figure Lengend Snippet: AID can process blunt DSBs to generate staggered DSBs. A 59 bp DNA fragment containing 13 RGYW repeats was cloned into pCR-Blunt II-TOPO ® vector. A 191 bp 5'-phosphorylated blunt-ended linearized DNA substrate and the dU contained control DNA substrate were generated by PCR amplification of the pCR-Blunt IITOPO® vector containing the 59 bp fragment DNA using the forward 5'-phosphorylated primer A or primer A1 (containing a dU instead of a dC at position 3), specific for the 5' region of the 59 bp DNA fragment and the reverse primer B, specific for the vector sequence 116 bp downstream of the inserted 59 bp DNA fragment. The linearized DNA substrate was incubated with nil or recombinant GST-mouse AID fusion protein, then treated with SAP and Antarctic Phosphatase. These catalyze the release of 5'-phosphate groups from DNA yielding non-phosphorylated DNA ends, which cannot been amplified by LM-PCR. Incubation of DNA pretreated with nil or AID with recombinant E. coli Ung, followed by treatment with Endo VIII showed that AID could process blunt DSBs to yield staggered DSB ends. Such staggered DSB ends were amplified by specific LM-PCR after treatment with T4 pol.

    Article Snippet: The thoroughly dephosphorylated DNA was treated with recombinant E. coli Ung (New England Biolabs Inc.) and then Endonuclease (Endo) VIII (New England Biolabs Inc.).

    Techniques: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Generated, Amplification, Sequencing, Incubation, Recombinant

    Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

    Journal: PLoS Genetics

    Article Title: ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

    doi: 10.1371/journal.pgen.1007605

    Figure Lengend Snippet: Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

    Article Snippet: For analysis of genomic and mitochondrial DNA composition by Endov-digestion and alkaline-gel electrophoresis, DNA samples were treated with 10 U of Endonuclease V (NEB) with the supplied buffer for 2 hours at 37°C.

    Techniques: Functional Assay, Derivative Assay, Nucleic Acid Electrophoresis, Migration, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Positive Control, Quantitation Assay

    Binding of ETS1 protein promotes UV damage formation in vitro.  a  DNA sequences of  RPL13A  and  SDHD  promoter fragments, corresponding to chromosome coordinates chr19:49990710-49990681 and chr11:111957515-111957553, respectively. Putative ETS motifs are shown in gray background and highlighted in bold. Recurrent mutated sites in melanomas are underlined.  b, c  Gel shift assays showing binding of purified ETS1 protein to radiolabeled  RPL13A  ( b ) and  SDHD  ( c ) promoter fragments, respectively.  d  A representative sequencing gel (15%) showing CPD formation in naked  RPL13A  (sample 1, with UV irradiation) and ETS1-bound  RPL13A  DNA (samples 2–5, UV irradiation). The binding products shown in part ( b ) were irradiated with 1KJ m −2  of UV-C light and CPD lesions were converted to single strand breaks by T4 endonuclease V digestion. The resulting DNA breaks were separated on a 15% denaturing sequencing gel to analyze damage abundance at different locations. A negative control (naked DNA without UV irradiation) was also digested with T4 endoV to show the background level of DNA cleavage in the absence of UV-induced DNA lesions. The first lane on the left shows a 10-nt DNA ladder.  e  Same as in part ( d ), except the  SDHD  promoter fragment was analyzed on a 12% gel. Asterisk indicates gel running artifact caused by bromophenol blue in the gel loading buffer. Both  RPL13A  and  SDHD  CPD formation experiments were conducted at least 3 times independently with consistent results

    Journal: Nature Communications

    Article Title: ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma

    doi: 10.1038/s41467-018-05064-0

    Figure Lengend Snippet: Binding of ETS1 protein promotes UV damage formation in vitro. a DNA sequences of RPL13A and SDHD promoter fragments, corresponding to chromosome coordinates chr19:49990710-49990681 and chr11:111957515-111957553, respectively. Putative ETS motifs are shown in gray background and highlighted in bold. Recurrent mutated sites in melanomas are underlined. b, c Gel shift assays showing binding of purified ETS1 protein to radiolabeled RPL13A ( b ) and SDHD ( c ) promoter fragments, respectively. d A representative sequencing gel (15%) showing CPD formation in naked RPL13A (sample 1, with UV irradiation) and ETS1-bound RPL13A DNA (samples 2–5, UV irradiation). The binding products shown in part ( b ) were irradiated with 1KJ m −2 of UV-C light and CPD lesions were converted to single strand breaks by T4 endonuclease V digestion. The resulting DNA breaks were separated on a 15% denaturing sequencing gel to analyze damage abundance at different locations. A negative control (naked DNA without UV irradiation) was also digested with T4 endoV to show the background level of DNA cleavage in the absence of UV-induced DNA lesions. The first lane on the left shows a 10-nt DNA ladder. e Same as in part ( d ), except the SDHD promoter fragment was analyzed on a 12% gel. Asterisk indicates gel running artifact caused by bromophenol blue in the gel loading buffer. Both RPL13A and SDHD CPD formation experiments were conducted at least 3 times independently with consistent results

    Article Snippet: The remaining DNA (i.e., 90%) was sequentially incubated with T4 endonuclease V and human apurinic/apyrimidinic (AP) endonuclease (APE1, NEB) to generate new ligatable 3′-OH groups at CPD sites.

    Techniques: Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Irradiation, Negative Control

    Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS.  a  Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced.  b  Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors  18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions.  c  Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions).  d  Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur.  e  Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS

    Journal: Nature Communications

    Article Title: ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma

    doi: 10.1038/s41467-018-05064-0

    Figure Lengend Snippet: Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS. a Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced. b Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors 18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions. c Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions). d Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur. e Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS

    Article Snippet: The remaining DNA (i.e., 90%) was sequentially incubated with T4 endonuclease V and human apurinic/apyrimidinic (AP) endonuclease (APE1, NEB) to generate new ligatable 3′-OH groups at CPD sites.

    Techniques: Genome Wide, Mutagenesis, Irradiation, Isolation, In Vitro

    SAMHD1 promoter is unmethylated in primary CD4 + T lymphocytes. A , genomic DNAs from the three donors' CD4 + T lymphocytes were either digested with HpaII (+) or left untreated (−) and subsequently subjected to PCR amplification using the P1/P2

    Journal: The Journal of Biological Chemistry

    Article Title: Promoter Methylation Regulates SAMHD1 Gene Expression in Human CD4+ T Cells *

    doi: 10.1074/jbc.M112.447201

    Figure Lengend Snippet: SAMHD1 promoter is unmethylated in primary CD4 + T lymphocytes. A , genomic DNAs from the three donors' CD4 + T lymphocytes were either digested with HpaII (+) or left untreated (−) and subsequently subjected to PCR amplification using the P1/P2

    Article Snippet: 2) For HpaII digestion of genomic DNA, followed by SAMHD1 promoter-specific PCR, genomic DNA (1 μg) was digested with 10 units of HpaII endonuclease (New England Biolabs) for 16 h at 37 °C.

    Techniques: Polymerase Chain Reaction, Amplification

    Quantitative RT-PCR results obtained for McrBC assay. McrBC is a methylation-specific endonuclease, which cleaves DNA containing 5′-methylcytosine residues but will not act on unmethylated DNA. Thus, a lesser PCR product recovery is indicative

    Journal: Molecular Endocrinology

    Article Title: Research Resource: Genome-Wide Profiling of Methylated Promoters in Endometriosis Reveals a Subtelomeric Location of Hypermethylation

    doi: 10.1210/me.2010-0160

    Figure Lengend Snippet: Quantitative RT-PCR results obtained for McrBC assay. McrBC is a methylation-specific endonuclease, which cleaves DNA containing 5′-methylcytosine residues but will not act on unmethylated DNA. Thus, a lesser PCR product recovery is indicative

    Article Snippet: DNA (1μg) was treated either with 25 U of McrBC endonuclease (New England Biolabs, Beverly, MA) or mock treated with an equivalent volume of water in a 50-μl reaction mixture containing 1× NEB2 buffer, 0.1 mg/ml BSA, and 2 m m GTP.

    Techniques: Quantitative RT-PCR, Methylation, Activated Clotting Time Assay, Polymerase Chain Reaction

    Electrophoresis on an agarose gel (0.8%, 40 V, 16 h) of HindIII restriction digests of 122-MDa plasmid DNA extracted from E. coli transconjugants of MDR serovar Typhi isolates. Lane 1, HindIII-digested bacteriophage lambda DNA. (A) Photograph of the agarose gel. (B) Image analyzed by computer.

    Journal: Journal of Clinical Microbiology

    Article Title: Endemic, Epidemic Clone of Salmonella enterica Serovar Typhi Harboring a Single Multidrug-Resistant Plasmid in Vietnam between 1995 and 2002

    doi: 10.1128/JCM.42.7.3094-3099.2004

    Figure Lengend Snippet: Electrophoresis on an agarose gel (0.8%, 40 V, 16 h) of HindIII restriction digests of 122-MDa plasmid DNA extracted from E. coli transconjugants of MDR serovar Typhi isolates. Lane 1, HindIII-digested bacteriophage lambda DNA. (A) Photograph of the agarose gel. (B) Image analyzed by computer.

    Article Snippet: Among the plasmids extracted from the previous 107 E. coli transconjugants, 54 plasmids (18 from the north, 14 from the center, and 22 from the south of Vietnam) of similar size were digested by EcoRI or HindIII endonuclease in ERB-lab, INHE, Hanoï, according to the manufacturer's instructions (New England Biolabs).

    Techniques: Electrophoresis, Agarose Gel Electrophoresis, Multiple Displacement Amplification, Plasmid Preparation, Lambda DNA Preparation

    Global DNA methylation and chromosomal accessibility in CD4 + T cells . A. - C . Genomic DNA isolated from total CD4 + T cells taken from the spleens of 2 week or 16 week old C57BL/6 mice that were either unstimulated (NS) or stimulated with PMA/I for 4 hours ( A ), FACS-sorted naive T cells from both age groups ( B ), or FACS-sorted naive T cells and nTreg cells or differentiated Th1, Th2, Th17, and iTreg cells ( C ) were digested with McrBC for 6 hours at 37°C and the DNA fragments were resolved on a 1% agarose gel. Uncut genomic DNA is also shown. D. E . Nuclei isolated from CD4 + T cells of 2 weeks ( D ) and 16 weeks ( E ) old C57BL/6 mice were digested with various concentrations of MNase (10 U to 120 U) at 37°C for 5 min, prior to loading onto a 1% agarose gel for separation. The DNA was visualised by ethidium bromide staining in all experiments. The 1 kb plus ladder is indicated by the letter L. Every experiment was repeated at least 3 times

    Journal: BMC Molecular Biology

    Article Title: Plasticity of DNA methylation in mouse T cell activation and differentiation

    doi: 10.1186/1471-2199-13-16

    Figure Lengend Snippet: Global DNA methylation and chromosomal accessibility in CD4 + T cells . A. - C . Genomic DNA isolated from total CD4 + T cells taken from the spleens of 2 week or 16 week old C57BL/6 mice that were either unstimulated (NS) or stimulated with PMA/I for 4 hours ( A ), FACS-sorted naive T cells from both age groups ( B ), or FACS-sorted naive T cells and nTreg cells or differentiated Th1, Th2, Th17, and iTreg cells ( C ) were digested with McrBC for 6 hours at 37°C and the DNA fragments were resolved on a 1% agarose gel. Uncut genomic DNA is also shown. D. E . Nuclei isolated from CD4 + T cells of 2 weeks ( D ) and 16 weeks ( E ) old C57BL/6 mice were digested with various concentrations of MNase (10 U to 120 U) at 37°C for 5 min, prior to loading onto a 1% agarose gel for separation. The DNA was visualised by ethidium bromide staining in all experiments. The 1 kb plus ladder is indicated by the letter L. Every experiment was repeated at least 3 times

    Article Snippet: Genomic DNA (1 μg) was digested with McrBC (10 units) endonuclease (NEB) in the presence of 10 mM GTP and NEBuffer 2.

    Techniques: DNA Methylation Assay, Isolation, Mouse Assay, FACS, Agarose Gel Electrophoresis, Staining