mspji  (New England Biolabs)


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    Name:
    MspJI
    Description:
    MspJI 1 000 units
    Catalog Number:
    R0661L
    Price:
    450
    Category:
    Restriction Enzymes
    Size:
    1 000 units
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    Structured Review

    New England Biolabs mspji
    MspJI
    MspJI 1 000 units
    https://www.bioz.com/result/mspji/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mspji - by Bioz Stars, 2021-05
    98/100 stars

    Images

    1) Product Images from "Heritable Epigenetic Variation among Maize Inbreds"

    Article Title: Heritable Epigenetic Variation among Maize Inbreds

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002372

    Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.
    Figure Legend Snippet: Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.

    Techniques Used: DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    2) Product Images from "Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9"

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

    Journal: mBio

    doi: 10.1128/mBio.00648-15

    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.
    Figure Legend 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.

    Techniques Used: Modification, Mobility Shift, Sequencing, Methylation

    3) Product Images from "Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI"

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    Journal: Genome Research

    doi: 10.1101/gr.222885.117

    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).
    Figure Legend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Techniques Used: Ligation, Fractionation, Amplification, Sequencing

    4) Product Images from "Degradation of non-methylated DNA by MsPJI impairs its usefulness as epigenetic tool"

    Article Title: Degradation of non-methylated DNA by MsPJI impairs its usefulness as epigenetic tool

    Journal: bioRxiv

    doi: 10.1101/072090

    Effect of MspJI on the amplification of non-methylated DNA. PCR product of 1799 bp was incubated in the absence (lane 1) or presence (lane 2) of MspJI. Treated samples were re-amplifiied by PCR. Positive (lane 3) and negative (lane 4) controls were included.
    Figure Legend Snippet: Effect of MspJI on the amplification of non-methylated DNA. PCR product of 1799 bp was incubated in the absence (lane 1) or presence (lane 2) of MspJI. Treated samples were re-amplifiied by PCR. Positive (lane 3) and negative (lane 4) controls were included.

    Techniques Used: Amplification, Methylation, Polymerase Chain Reaction, Incubation

    Effect of MspJI treatment on the real time amplification of genomic templates. qPCR reactions were performed using murine genomic DNA digested (D) or not digested (ND) with MspJI. The target sequences contained 14 (A) or none (B) CpG sites. The curves are representative of 12 independent experiments performed in duplicates.
    Figure Legend Snippet: Effect of MspJI treatment on the real time amplification of genomic templates. qPCR reactions were performed using murine genomic DNA digested (D) or not digested (ND) with MspJI. The target sequences contained 14 (A) or none (B) CpG sites. The curves are representative of 12 independent experiments performed in duplicates.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction

    PCR amplification of IL-12b promoter gene. Genomic DNA was incubated in the absence (lanes 2 and 3) or the presence (lanes 4 and 5) of MspJI, and used as template. Positive (lane 6) and negative (lane 7) amplification controls were analyzed in parallel.
    Figure Legend Snippet: PCR amplification of IL-12b promoter gene. Genomic DNA was incubated in the absence (lanes 2 and 3) or the presence (lanes 4 and 5) of MspJI, and used as template. Positive (lane 6) and negative (lane 7) amplification controls were analyzed in parallel.

    Techniques Used: Polymerase Chain Reaction, Amplification, Incubation

    Schematic representation of the experimental design. Genomic DNA is treated with MspJI. If DNA molecules are non-methylated (left), template remains intact and can be amplified by PCR. If DNA is methylated (right), MspJI degrades template and weaker or no amplification take place. Partial methylation can be better detected by qPCR as a shift in Cq values.
    Figure Legend Snippet: Schematic representation of the experimental design. Genomic DNA is treated with MspJI. If DNA molecules are non-methylated (left), template remains intact and can be amplified by PCR. If DNA is methylated (right), MspJI degrades template and weaker or no amplification take place. Partial methylation can be better detected by qPCR as a shift in Cq values.

    Techniques Used: Methylation, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Electrophoresis of genomic DNA from M. musculus or G. lamblia digested with MspJI. Lane 1: 1kb DNA ladder; lane 2 and 5: undigested DNA; lanes 3 and 6: DNA incubated with MspJI for 4 hs; lane 4: DNA incubated with MspJI for 8 hs.
    Figure Legend Snippet: Electrophoresis of genomic DNA from M. musculus or G. lamblia digested with MspJI. Lane 1: 1kb DNA ladder; lane 2 and 5: undigested DNA; lanes 3 and 6: DNA incubated with MspJI for 4 hs; lane 4: DNA incubated with MspJI for 8 hs.

    Techniques Used: Electrophoresis, Incubation

    Related Articles

    Incubation:

    Article Title: Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development
    Article Snippet: .. After spinning down the 384-well plates, single cells are deposited into each well of the plate and incubated at 50 °C for 15 h, 75 °C for 20 min, and 80 °C for 5 min. 5hmC sites in the genome are then glucosylated to block downstream recognition by MspJI by dispensing 0.5 μL of the following reaction mixture: 0.1 μL of T4-BGT (NEB), 0.1 μL of UDP-Glucose (NEB), 0.05 μL of 10× NEB Buffer 4, and 0.25 μL of nuclease-free water. .. After incubation at 37 °C for 16 h, 0.5 μL the following reaction mixture is added: 0.1 μL of 25 μg/μL Qiagen Protease, 0.05 μL of 10× NEB Buffer 4, and 0.35 μL of nuclease-free water.

    Article Title: Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria
    Article Snippet: .. The genomic DNA (1000 ng) was digested for 15 min using 2.5 units of Msp JI (NEB, Ipswich, MA, USA), McrBC (NEB), Eco RII (Thermo Scientific), and Bsp119I (Thermo Scientific) at the optimal incubation temperature for each enzyme. ..

    Blocking Assay:

    Article Title: Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development
    Article Snippet: .. After spinning down the 384-well plates, single cells are deposited into each well of the plate and incubated at 50 °C for 15 h, 75 °C for 20 min, and 80 °C for 5 min. 5hmC sites in the genome are then glucosylated to block downstream recognition by MspJI by dispensing 0.5 μL of the following reaction mixture: 0.1 μL of T4-BGT (NEB), 0.1 μL of UDP-Glucose (NEB), 0.05 μL of 10× NEB Buffer 4, and 0.25 μL of nuclease-free water. .. After incubation at 37 °C for 16 h, 0.5 μL the following reaction mixture is added: 0.1 μL of 25 μg/μL Qiagen Protease, 0.05 μL of 10× NEB Buffer 4, and 0.35 μL of nuclease-free water.

    other:

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI
    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

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

    Methylation:

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: PCR following treatment with a methylation-dependent restriction enzyme (PTMR) PTMR was performed according to the method reported by Shigematsu et al. . .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Sequencing:

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: PCR following treatment with a methylation-dependent restriction enzyme (PTMR) PTMR was performed according to the method reported by Shigematsu et al. . .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

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    New England Biolabs mspji
    Variable <t>DNA</t> methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme <t>MspjI</t> followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.
    Mspji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mspji/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mspji - by Bioz Stars, 2021-05
    98/100 stars
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    Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.

    Journal: PLoS Genetics

    Article Title: Heritable Epigenetic Variation among Maize Inbreds

    doi: 10.1371/journal.pgen.1002372

    Figure Lengend Snippet: Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.

    Article Snippet: 1 microgram of genomic DNA was digested for 16 hr with MspJI or FspEI (New England Biolabs).

    Techniques: DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative

    Journal: Acta neuropathologica

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

    doi: 10.1007/s00401-014-1286-y

    Figure Lengend Snippet: Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative

    Article Snippet: DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction.

    Techniques: Polymerase Chain Reaction

    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

    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Journal: Genome Research

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    doi: 10.1101/gr.222885.117

    Figure Lengend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

    Techniques: Ligation, Fractionation, Amplification, Sequencing