t4 β glucosyltransferase  (New England Biolabs)


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  • 99
    Name:
    T4 Phage beta glucosyltransferase
    Description:
    T4 Phage beta glucosyltransferase 2 500 units
    Catalog Number:
    M0357L
    Price:
    351
    Size:
    2 500 units
    Category:
    Other Enzymes
    Score:
    85
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    Structured Review

    New England Biolabs t4 β glucosyltransferase
    T4 Phage beta glucosyltransferase
    T4 Phage beta glucosyltransferase 2 500 units
    https://www.bioz.com/result/t4 β glucosyltransferase/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    t4 β glucosyltransferase - by Bioz Stars, 2019-10
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C. .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Amplification:

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB). .. For the “C” or “C + mC + hmC” libraries, the aliquots of DNA without glucosylation were directly digested with HpaII or MspI (NEB), respectively.

    Filtration:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Synthesized:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Quantitative RT-PCR:

    Article Title: Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus
    Article Snippet: The 5hmC modifications of 5-LOX DNA sequences were quantified using an assay that involves a 5hmC-sensitive enzymatic restriction digest combined with glucosylation and qRT-PCR. .. Prior to restriction digestion, DNA was glucosylated with T4 glucosyl transferase (New England Biolabs) and aliquots were digested with the indicated enzymes in separate reactions.

    Real-time Polymerase Chain Reaction:

    Article Title: DNA methylation and differentiation: HOX genes in muscle cells
    Article Snippet: Because Mb and Mt have particularly high levels of the RNA encoding TET1 and TET2, enzymes that generate 5hmC from 5mC residues [ ], and RRBS cannot distinguish between 5hmC and 5mC [ ], it was important to determine relative amounts of these modified C residues at representative HOX cluster sites. .. We quantified 5mC and 5hmC at a MbMt-hypermethylated Msp I site (5′-CCGG-3′) in the single introns of HOXB5 and HOXD4 , exon 1 of HOXA5 , exon 2 of HOXC6 , and 1.7 kb upstream of the TSS of HOXA7 (Figures , , and ) by an enzymatic assay that involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark; New England Biolabs, Ipswich, MA, USA), digestion with Msp I or Hpa II and real-time PCR [ ]. .. Using sets of samples independent from those for RRBS, the hypermethylation of these five sites in Mb and of the HOXD4 and HOXA7 sites in skeletal muscle was verified by this assay (Table ).

    Article Title: FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency
    Article Snippet: Briefly, 250 ng of genomic DNA was treated with T4 Phage β-glucosyltransferase (NEB M0357S) according to the manufacturer’s instructions. .. Seventy nanograms of glucosylated gDNA were digested with 10 U of HpaII or MspI or no enzyme (mock digestion) at 37°C for 2 hr, then inactivated for 20 min at 80°C.

    Article Title: Myogenic Differential Methylation: Diverse Associations with Chromatin Structure
    Article Snippet: In several types of DNA samples, we examined the CCGG site 406 bp downstream of the MARVELD2 TSS and residing within the myogenic DMR. .. The assay involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark, New England Biolabs), digestion with MspI or HpaII, and real-time PCR [ ]. .. The results confirmed that Mb contained much higher amounts of 5mC at the examined site than did skin fibroblasts (average of three samples each, 95 and 35%, respectively, with < 0.01% detectable 5hmC).

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects
    Article Snippet: We utilized glucosylation of genomic DNA followed by methylation-sensitive qPCR (glucMS-qPCR) to quantify levels of 5-methyl-cytosine and 5-hydroxy-methyl-cytosine. .. Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight.

    Article Title: Early de novo DNA methylation and prolonged demethylation in the muscle lineage
    Article Snippet: For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described. .. For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described.

    Article Title: Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells
    Article Snippet: These digested DNAs ware subjected to quantitative PCR (qPCR) analysis using primers designed for the Insulin-like growth factor 1 (Igf1) promoter. .. To determine the level of 5hmC, we sequentially treated the genomic DNA with MspI and T4-β-glucosyltransferase (BGT, New England Biolabs, USA) following the manufacturer’s instructions.

    Article Title: Effect of valproic acid on mitochondrial epigenetics
    Article Snippet: The sequence-specific 5hmC content in selected mtDNA regions was measured by qPCR applied to samples after a glucosyltransferase step followed by an enzymatic restriction digest with two enzymes, CviAII (CATG recognition site) and CviQI (GTAC recognition site) (New England Biolabs; Ipswich, MA) ( ). .. First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues.

    Article Title: MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors
    Article Snippet: Paragraph title: Quantification of 5hmC Levels in gDNA by Methylation-Sensitive qPCR ... gDNA was incubated with T4 Phage β-glucosyltransferase (New England Biolabs, Ipswich, MA) by following the manufacturer’s protocol.

    Incubation:

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects
    Article Snippet: Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight. .. Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight.

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: A 900 ng portion of high-molecular-weight DNA from PBMCs was subjected to a nick-translation reaction with Nt.BspQI for nicking and atto-532-dUTP for labeling. .. For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume). .. Next, dibenzocyclooctyl (DBCO)-Cy5 (Jena Bioscience) was added to a final concentration of 620 μM and the reaction was incubated overnight at 37 °C.

    Article Title: Early de novo DNA methylation and prolonged demethylation in the muscle lineage
    Article Snippet: In addition, we used previously unreported data from our expression profiling of Mb and Mt vs. 19 types of non-muscle cell cultures on microarrays (Affymetrix Exon 1.0 ST ). .. For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described. .. All glucosylation reaction values were corrected for non-specific binding of UDP-[3 H]glucose to membranes using a DNA-minus reaction.

    Article Title: MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors
    Article Snippet: Relative fractions of methylated and unmethylated DNA were measured by comparing the amount in each digest with that of the mock digest using the ΔCt method. .. gDNA was incubated with T4 Phage β-glucosyltransferase (New England Biolabs, Ipswich, MA) by following the manufacturer’s protocol. .. First, 100 ng of glucosylated gDNA was digested with HpaII, MspI, or without enzyme (mock) at 37°C overnight and then incubated for 20 minutes at 80°C for enzyme deactivation.

    Expressing:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Modification:

    Article Title: DNA methylation and differentiation: HOX genes in muscle cells
    Article Snippet: Because Mb and Mt have particularly high levels of the RNA encoding TET1 and TET2, enzymes that generate 5hmC from 5mC residues [ ], and RRBS cannot distinguish between 5hmC and 5mC [ ], it was important to determine relative amounts of these modified C residues at representative HOX cluster sites. .. We quantified 5mC and 5hmC at a MbMt-hypermethylated Msp I site (5′-CCGG-3′) in the single introns of HOXB5 and HOXD4 , exon 1 of HOXA5 , exon 2 of HOXC6 , and 1.7 kb upstream of the TSS of HOXA7 (Figures , , and ) by an enzymatic assay that involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark; New England Biolabs, Ipswich, MA, USA), digestion with Msp I or Hpa II and real-time PCR [ ].

    Article Title: Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus
    Article Snippet: Prior to restriction digestion, DNA was glucosylated with T4 glucosyl transferase (New England Biolabs) and aliquots were digested with the indicated enzymes in separate reactions. .. Prior to restriction digestion, DNA was glucosylated with T4 glucosyl transferase (New England Biolabs) and aliquots were digested with the indicated enzymes in separate reactions.

    Article Title: Effect of valproic acid on mitochondrial epigenetics
    Article Snippet: First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues. .. First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues.

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine
    Article Snippet: PCRs were carried out using Phusion polymerase (NEB #M0530). .. The DNA fragment containing 5ghmC was obtained by further modification of 5hmC DNA fragment by the T4 β-glucosyltransferase (NEB #M0357). .. All PCR primers are listed in the Supplementary Table S2 .

    Ligation:

    Article Title: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells
    Article Snippet: Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4. .. The AbaSI digested DNA was then purified by phenol-chloroform extraction and ethanol precipitation.

    Nick Translation:

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: A 900 ng portion of high-molecular-weight DNA from PBMCs was subjected to a nick-translation reaction with Nt.BspQI for nicking and atto-532-dUTP for labeling. .. For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume).

    Cell Culture:

    Article Title: Myogenic Differential Methylation: Diverse Associations with Chromatin Structure
    Article Snippet: The assay involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark, New England Biolabs), digestion with MspI or HpaII, and real-time PCR [ ]. .. The assay involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark, New England Biolabs), digestion with MspI or HpaII, and real-time PCR [ ].

    Article Title: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells
    Article Snippet: The undifferentiated E14 cells was cultured as previously described ( ). .. Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4.

    other:

    Article Title:
    Article Snippet: The hmC residues within the MspI site were glucosylated by incubating 200 pmol of DNA substrates with 1 μl (10 units) of T4 β-glucosyltransferase (β-GT) (NEB) for 1 h at 37 °C in a total 50-μl reaction containing 1× NEBuffer 4 supplemented with 0.1 mm UDP-Glc.

    Sequencing:

    Article Title: Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus
    Article Snippet: Paragraph title: 2.6. Sequence-specific DNA 5hmC assay ... Prior to restriction digestion, DNA was glucosylated with T4 glucosyl transferase (New England Biolabs) and aliquots were digested with the indicated enzymes in separate reactions.

    Article Title: Gene-body 5-hydroxymethylation is associated with gene expression changes in the prefrontal cortex of depressed individuals
    Article Snippet: Purity and concentrations of genomic DNA were assessed using the Thermo Scientific Nanodrop 1000 spectrophotometer (Carlsbad, CA, USA) and each sample had a 260/280 ratio > 1.8. .. AbaSI sequencing library construction was performed as described previously., Briefly, DNA was glucosylated by using UDP-glucose and T4-β-glucosyltransferase (New England Biolabs, Ipswich, MA, USA, cat. #M0357L). .. The DNA was then digested using AbaSI (New England Biolabs, cat. #R0665S) and custom-biotinylated adaptors were ligated to DNA ends.

    Article Title: Effect of valproic acid on mitochondrial epigenetics
    Article Snippet: Paragraph title: 2.5. mtDNA sequence-specific 5hmC assay ... First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues.

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: Paragraph title: Data analysis by hydroxymethylation and methylation sensitive tag sequencing (HMST-Seq) ... For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB).

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine
    Article Snippet: The DNA fragment containing 5ghmC was obtained by further modification of 5hmC DNA fragment by the T4 β-glucosyltransferase (NEB #M0357). .. The synthetic oligonucleotides used in were made from PCR by using primers hmCG_ACGT_F and hmCG_ACGT_R on the hmCG_ACGT_template in the presence of dhmCTP /dATP/dGTP/dTTP ( Supplementary Table S3 ).

    Recombinant:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Staining:

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume). .. For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume).

    Radioactivity:

    Article Title: Early de novo DNA methylation and prolonged demethylation in the muscle lineage
    Article Snippet: In addition, we used previously unreported data from our expression profiling of Mb and Mt vs. 19 types of non-muscle cell cultures on microarrays (Affymetrix Exon 1.0 ST ). .. For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described. .. All glucosylation reaction values were corrected for non-specific binding of UDP-[3 H]glucose to membranes using a DNA-minus reaction.

    RNA Sequencing Assay:

    Article Title: Myogenic Differential Methylation: Diverse Associations with Chromatin Structure
    Article Snippet: MARVELD2 is strongly repressed in Mb and Mt ( c and , ENCODE/Strand-specific RNA-seq, long RNA-seq, > 200 nt poly(A)+ RNA, Tom Gingeras, Cold Spring Harbor Laboratory, and ENCODE/RNA-seq, > 200 nt poly(A)+ RNA, Barbara Wold, California Institute of Technology) and displays a hypermethylated DMR in Mb and Mt spanning from immediately upstream to downstream of the TSS ( a,d). .. The assay involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark, New England Biolabs), digestion with MspI or HpaII, and real-time PCR [ ].

    DNA Purification:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Methylation:

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects
    Article Snippet: We utilized glucosylation of genomic DNA followed by methylation-sensitive qPCR (glucMS-qPCR) to quantify levels of 5-methyl-cytosine and 5-hydroxy-methyl-cytosine. .. Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight.

    Article Title: Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus
    Article Snippet: The following enzymes were selected based on their published characteristics (REBASE Methylation Sensitivity: ) and their recognition sequences are indicated in parentheses: TseI [GCWGC], NmeAIII [GCCGAG (21/19)], EciI [GGCGGA (11/9)], SfaNI [GCATC (5/9)], and EcoP15I [CAGCAG (25/27)] (New England Biolabs; Ipswich, MA). .. Prior to restriction digestion, DNA was glucosylated with T4 glucosyl transferase (New England Biolabs) and aliquots were digested with the indicated enzymes in separate reactions.

    Article Title: Early de novo DNA methylation and prolonged demethylation in the muscle lineage
    Article Snippet: For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described. .. For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described.

    Article Title: Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells
    Article Snippet: 2.4 For the measurement of DNA methylation state, the genomic DNA was digested with either MspI (that recognized and cleaved CCGG sequences independent of its methylation) or HapII (that recognized the same sequences as MspI but could not cleave methylated DNA). .. To determine the level of 5hmC, we sequentially treated the genomic DNA with MspI and T4-β-glucosyltransferase (BGT, New England Biolabs, USA) following the manufacturer’s instructions.

    Article Title: Effect of valproic acid on mitochondrial epigenetics
    Article Snippet: First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues. .. First, DNA samples are glucosylated with phage β-glucosyltransferase (T4-BGT; New England Biolabs, Ipswich, MA), which specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5hmC residues but not to the 5mC residues.

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: Paragraph title: Data analysis by hydroxymethylation and methylation sensitive tag sequencing (HMST-Seq) ... For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB).

    Article Title: MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors
    Article Snippet: Paragraph title: Quantification of 5hmC Levels in gDNA by Methylation-Sensitive qPCR ... gDNA was incubated with T4 Phage β-glucosyltransferase (New England Biolabs, Ipswich, MA) by following the manufacturer’s protocol.

    Isolation:

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects
    Article Snippet: Genomic DNA was isolated from treated neurospheres using the DNeasy Blood and Tissue kit (Cat# 69506; QIAGEN) according to the recommended protocol. .. Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight.

    Labeling:

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: A 900 ng portion of high-molecular-weight DNA from PBMCs was subjected to a nick-translation reaction with Nt.BspQI for nicking and atto-532-dUTP for labeling. .. For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume). .. Next, dibenzocyclooctyl (DBCO)-Cy5 (Jena Bioscience) was added to a final concentration of 620 μM and the reaction was incubated overnight at 37 °C.

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine
    Article Snippet: The DNA fragment containing 5ghmC was obtained by further modification of 5hmC DNA fragment by the T4 β-glucosyltransferase (NEB #M0357). .. The oligonucleotide sequence is designed so that there is only one CG site (underlined in Supplementary Table S3 ), which contains the 5hmC in the top strand.

    Purification:

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume). .. For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume).

    Article Title: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells
    Article Snippet: Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4. .. Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4.

    Article Title: Early de novo DNA methylation and prolonged demethylation in the muscle lineage
    Article Snippet: In addition, we used previously unreported data from our expression profiling of Mb and Mt vs. 19 types of non-muscle cell cultures on microarrays (Affymetrix Exon 1.0 ST ). .. For the determination of the levels of genomic 5-hmC, a T4 β-glucosyltransferase (β-GT; New England Biolabs) glucosylation assay was performed on 2 µg of purified genomic DNA incubated with UDP-[3 H]glucose and β-GT and applied to a DE81 membrane with determination of the membrane-bound radioactivity, as previously described. .. All glucosylation reaction values were corrected for non-specific binding of UDP-[3 H]glucose to membranes using a DNA-minus reaction.

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: CAUTION Ethidium bromide is toxic and suspected of causing genetic defects if inhaled; use personal protective equipment when handling. .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C. .. Fe(NH4 )2 (SO4 )2 ·6H2 O (Alfa Aesar, cat. no. 13448; dissolved in H2 O at 1.5 mM) !

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB). .. After digestion, the digested aliquots of DNA from all three libraries were ligated with biotinylated linker, fragmented by NlaIII, captured by streptavidin-conjugated beads, digested with MmeI to generate short sequence tags (16–17 bp), and ligated with sequencing linkers and amplified by PCR.

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine
    Article Snippet: The DNA fragment containing 5ghmC was obtained by further modification of 5hmC DNA fragment by the T4 β-glucosyltransferase (NEB #M0357). .. The oligonucleotide sequence is designed so that there is only one CG site (underlined in Supplementary Table S3 ), which contains the 5hmC in the top strand.

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Polymerase Chain Reaction:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: CAUTION Ethidium bromide is toxic and suspected of causing genetic defects if inhaled; use personal protective equipment when handling. .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C. .. Fe(NH4 )2 (SO4 )2 ·6H2 O (Alfa Aesar, cat. no. 13448; dissolved in H2 O at 1.5 mM) !

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB). .. For the “C” or “C + mC + hmC” libraries, the aliquots of DNA without glucosylation were directly digested with HpaII or MspI (NEB), respectively.

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine
    Article Snippet: To prepare the DNA substrates used in C and , DNA fragments were PCR-amplified from the T4 gt genomic or pUC19 DNA by using dATP/dGTP/dTTP mixed with dhmCTP (Bioline #BIO-39046), dmCTP (NEB #N0356S) or dCTP, respectively. .. The DNA fragment containing 5ghmC was obtained by further modification of 5hmC DNA fragment by the T4 β-glucosyltransferase (NEB #M0357).

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Construct:

    Article Title: Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue
    Article Snippet: Three independent libraries with initially different enzyme digestion were constructed for each sample, which were termed “C” library (c), “C + mC” library (a) and “C + mC + hmC” (b), respectively. .. For “C + mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating DNA substrates with T4 β-glucosyltransferase (β-GT) (NEB).

    Lysis:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Plasmid Preparation:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Ethanol Precipitation:

    Article Title: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine in Mouse Embryonic Stem Cells
    Article Snippet: Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4. .. Genomic DNA was glucosylated overnight at 37°C by incubating 100 ng of E14 genomic DNA with 200 μM UDP-glucose and 20 units of T4-β-glucosyl-transferase (NEB: M0357S) in NEB buffer #4.

    Enzymatic Assay:

    Article Title: DNA methylation and differentiation: HOX genes in muscle cells
    Article Snippet: Because Mb and Mt have particularly high levels of the RNA encoding TET1 and TET2, enzymes that generate 5hmC from 5mC residues [ ], and RRBS cannot distinguish between 5hmC and 5mC [ ], it was important to determine relative amounts of these modified C residues at representative HOX cluster sites. .. We quantified 5mC and 5hmC at a MbMt-hypermethylated Msp I site (5′-CCGG-3′) in the single introns of HOXB5 and HOXD4 , exon 1 of HOXA5 , exon 2 of HOXC6 , and 1.7 kb upstream of the TSS of HOXA7 (Figures , , and ) by an enzymatic assay that involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark; New England Biolabs, Ipswich, MA, USA), digestion with Msp I or Hpa II and real-time PCR [ ]. .. Using sets of samples independent from those for RRBS, the hypermethylation of these five sites in Mb and of the HOXD4 and HOXA7 sites in skeletal muscle was verified by this assay (Table ).

    Article Title: Myogenic Differential Methylation: Diverse Associations with Chromatin Structure
    Article Snippet: Because 5-hydroxymethylcytosine (5hmC) can be mistaken for 5-methylcytosine (5mC) by RRBS, we used an enzymatic assay to ensure that we are correctly interpreting RRBS signal as 5mC. .. The assay involves glucosylation of 5hmC by T4 phage β-glucosyltransferase (β-GT; Epimark, New England Biolabs), digestion with MspI or HpaII, and real-time PCR [ ].

    Spectrophotometry:

    Article Title: Characterizing 5-hydroxymethylcytosine in human prefrontal cortex at single base resolution
    Article Snippet: Concentrations of genomic DNA were assessed using the Thermo Scientific Nanodrop 1000 spectrophotometer and each sample had a 260/280 ratio greater than 1.8. .. Briefly, DNA was glucosylated by using UDP-glucose and T4-β-glucosyltransferase (New England Biolabs, cat. #M0357L).

    Article Title: Gene-body 5-hydroxymethylation is associated with gene expression changes in the prefrontal cortex of depressed individuals
    Article Snippet: Purity and concentrations of genomic DNA were assessed using the Thermo Scientific Nanodrop 1000 spectrophotometer (Carlsbad, CA, USA) and each sample had a 260/280 ratio > 1.8. .. AbaSI sequencing library construction was performed as described previously., Briefly, DNA was glucosylated by using UDP-glucose and T4-β-glucosyltransferase (New England Biolabs, Ipswich, MA, USA, cat. #M0357L).

    DNA Methylation Assay:

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects
    Article Snippet: Paragraph title: Analysis of DNA methylation and DNA hydroxymethylation ... Here, 30 µg of genomic DNA was treated with 30 units of T4 phage β-glucosyltransferase (T4 BGT, Cat# M0357S; NEB) at 37 °C overnight.

    Article Title: Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells
    Article Snippet: Paragraph title: DNA methylation analysis using restriction enzymes and glucosyltransferase ... To determine the level of 5hmC, we sequentially treated the genomic DNA with MspI and T4-β-glucosyltransferase (BGT, New England Biolabs, USA) following the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
    Article Snippet: For labeling of 5-hmC, nick-labeled DNA was incubated overnight at 37 °C with 90 units of T4 β-glucosyltransferase (New England Biolabs), in the presence of 13.5 μL of 10× buffer 4 (New England Biolabs), 200 mM UDP-glucose-azide, and ultrapure water (135 μL total reaction volume). .. Next, dibenzocyclooctyl (DBCO)-Cy5 (Jena Bioscience) was added to a final concentration of 620 μM and the reaction was incubated overnight at 37 °C.

    BAC Assay:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: Genomic DNA sample (3 μg of genomic DNA purified with a genomic DNA purification kit (Wizard, cat. no. A1120 or similar)) Milli-Q water Recombinant mTet1 (3 mg ml − 1 ; Box 1 for expression and purification procedures) pFastBac dual-mTet1 (available on request from the corresponding author; sequence in ) Bac-to-Bac baculovirus expression system (Invitrogen, cat. no. 10359-016) High Five cells (Invitrogen, cat. no. B855-02) Grace's insect medium, supplemented (Invitrogen, cat. no. 11605102) Medium for mTet1 expression (Reagent Setup) Penicillin-streptomycin, liquid (Invitrogen, cat. no. 15140-163) FBS (Gemini Bio, cat. no. GBP-900198) Tris hydrochloride (Tris-HCl; 20 mM, pH 8.0; Fisher BioReagents, cat. no. BP1531) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) Phenylmethylsulfonyl fluoride (PMSF; Chem-impex international, cat. no. 00632) Leupeptin hemisulfate salt (Sigma-Aldrich, cat. no. L2884) Pepstatin A (Fisher BioReagents, cat. no. BP2671-100) Anti-flag M2 affinity gel (Sigma-Aldrich, cat. no. A2220) 3× Flag peptide (Sigma-Aldrich, cat. no. F4799; dissolved in 150 mM NaCl at 0.2 mg ml − 1 ) mTet1 cell lysis buffer (Reagent Setup) mTet1 gel filtration running buffer (Reagent Setup) Unmethylated λ-DNA (Promega, cat. no. D1521) SssI CpG methyltransferase (NEB, cat. no. M0226M; kit contains NEBuffer 2 and SAM) QIAEX II gel extraction kit (Qiagen, cat. no. 20021) ZymoTaq DNA polymerase (Zymo Research, cat. no. E2001) Hydroxymethyl dCTP (hmdCTP; Bioline, cat. no. BIO-39046) Ultrapure dATP (USB, cat. no. 77102) Ultrapure dGTP (USB, cat. no. 77106) Ultrapure dTTP (USB, cat. no. 77108) 5-hmC dNTP mix, 10 mM (Reagent Setup) 5-hmC_F_1, 5-hmC_R_1, 5-hmC_F_2, and 5-hmC_R_2 primers (synthesized by Operon; ) pUC 19 vector (NEB, cat. no. N3041S; dilute in H2 O to 1 ng μl − 1 ) Agarose (Denville, cat. no. CA3510-8) Ethidium bromide (Acros, cat. no. 170960050; dissolved in H2 O at 10 mg ml − 1 ) ! .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C.

    Gel Extraction:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: CAUTION Ethidium bromide is toxic and suspected of causing genetic defects if inhaled; use personal protective equipment when handling. .. QIAquick PCR purification kit (Qiagen, cat. no. 28104) MinElute gel extraction kit (Qiagen, cat. no. 28004) HEPES (500 mM and 1 M, pH 8.0; GenScript, cat. no. ) MgCl2 ·6H2 O (Fluka, cat. no. 63064; dissolved in H2 O at 250 mM) β-GT protection buffer 10× (Reagent Setup) T4 Phage β-GT (T4-βGT; NEB, cat. no. M0357S) UDP-glucose (Wako, cat. no. EPL1144; dissolved in H2 O at 10 mM) ▲ CRITICAL UDP-glucose should be stored at − 20 °C. .. Fe(NH4 )2 (SO4 )2 ·6H2 O (Alfa Aesar, cat. no. 13448; dissolved in H2 O at 1.5 mM) !

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Hood:

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500)

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    New England Biolabs t4 β glucosyltransferase
    T4 β Glucosyltransferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 β glucosyltransferase/product/New England Biolabs
    Average 97 stars, based on 5 article reviews
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    t4 β glucosyltransferase - by Bioz Stars, 2019-10
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    90
    New England Biolabs t4 phage beta glucosyltransferase
    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 <t>glucosyltransferase</t> (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    T4 Phage Beta Glucosyltransferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 phage beta glucosyltransferase/product/New England Biolabs
    Average 90 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    t4 phage beta glucosyltransferase - by Bioz Stars, 2019-10
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    Image Search Results


    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.217083

    Figure Lengend Snippet: Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.

    Article Snippet: The hmC residues within the MspI site were glucosylated by incubating 200 pmol of DNA substrates with 1 μl (10 units) of T4 β-glucosyltransferase (β-GT) (NEB) for 1 h at 37 °C in a total 50-μl reaction containing 1× NEBuffer 4 supplemented with 0.1 mm UDP-Glc.

    Techniques: Quantitation Assay, Methylation, Gas Chromatography, Real-time Polymerase Chain Reaction