monarch genomic dna purification kit  (New England Biolabs)


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    New England Biolabs monarch genomic dna purification kit
    Monarch Genomic Dna Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    monarch genomic dna purification kits  (New England Biolabs)


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    New England Biolabs monarch genomic dna purification kits
    Monarch Genomic Dna Purification Kits, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch genomic dna purification kits/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch genomic dna purification kits - by Bioz Stars, 2023-01
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    monarch genomic dna purification kit  (New England Biolabs)


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    New England Biolabs monarch genomic dna purification kit
    A) The 6mA-2-thio-dTTP base pair forms weaker hydrogen bonds compared to dA-dTTP and dA-2-thio-dTTP base pairs. B) Heatmap plot for the 6mA-to-T/C/G mutation ratios at 256 motifs (NN-6mA-NN) on the single-stranded model <t>DNA</t> after mutation-prone primer extension. C) The mutation patterns (6mA to T, C, and G respectively) at 256 motifs (NN-6mA-NN) on the single-stranded model DNA. D) FTO demethylates over 99% methylated base of NN-6mA-NN model ssDNA in vitro and shows weaker activity on dsDNA. Data are mean ± s.e.m.; analyzed by two-tailed unpaired t-tests. The number of independently repeated reactions are shown in each plot. **** P <0.0001. E) Schematic diagram of DR-6mA-seq. <t>Genomic</t> <t>DNA</t> is sequentially denatured, mixed with spike-in probes, treated with FTO (or not), ligated to biotin-tagged adaptors, and extended by Bst 2.0 DNA polymerase or Q5 high-fidelity DNA polymerase. The synthesized DNA strand is enriched and amplified for next-generation sequencing. 6mA-to-T/C/G mutations were counted for defining 6mA sites and calculating 6mA methylation stoichiometry, using spike-in calibration curves. F) Mutation profiles (including 6mA to T, C, and G) of mutation-prone primer extension on the 6mA spike-in probes harboring 0%, 25%, 50%, 75% and 100% 6mA. Primer extension on FTO-treated probes and primer extension by Q5 high-fidelity DNA polymerase serve as two control groups and do not respond to 6mA fractions. See also Table S1 .
    Monarch Genomic Dna Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch genomic dna purification kit/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch genomic dna purification kit - by Bioz Stars, 2023-01
    86/100 stars

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    1) Product Images from "Sequencing of N 6 -methyl-deoxyadenosine at single-base resolution across the mammalian genome"

    Article Title: Sequencing of N 6 -methyl-deoxyadenosine at single-base resolution across the mammalian genome

    Journal: bioRxiv

    doi: 10.1101/2023.01.16.524325

    A) The 6mA-2-thio-dTTP base pair forms weaker hydrogen bonds compared to dA-dTTP and dA-2-thio-dTTP base pairs. B) Heatmap plot for the 6mA-to-T/C/G mutation ratios at 256 motifs (NN-6mA-NN) on the single-stranded model DNA after mutation-prone primer extension. C) The mutation patterns (6mA to T, C, and G respectively) at 256 motifs (NN-6mA-NN) on the single-stranded model DNA. D) FTO demethylates over 99% methylated base of NN-6mA-NN model ssDNA in vitro and shows weaker activity on dsDNA. Data are mean ± s.e.m.; analyzed by two-tailed unpaired t-tests. The number of independently repeated reactions are shown in each plot. **** P <0.0001. E) Schematic diagram of DR-6mA-seq. Genomic DNA is sequentially denatured, mixed with spike-in probes, treated with FTO (or not), ligated to biotin-tagged adaptors, and extended by Bst 2.0 DNA polymerase or Q5 high-fidelity DNA polymerase. The synthesized DNA strand is enriched and amplified for next-generation sequencing. 6mA-to-T/C/G mutations were counted for defining 6mA sites and calculating 6mA methylation stoichiometry, using spike-in calibration curves. F) Mutation profiles (including 6mA to T, C, and G) of mutation-prone primer extension on the 6mA spike-in probes harboring 0%, 25%, 50%, 75% and 100% 6mA. Primer extension on FTO-treated probes and primer extension by Q5 high-fidelity DNA polymerase serve as two control groups and do not respond to 6mA fractions. See also Table S1 .
    Figure Legend Snippet: A) The 6mA-2-thio-dTTP base pair forms weaker hydrogen bonds compared to dA-dTTP and dA-2-thio-dTTP base pairs. B) Heatmap plot for the 6mA-to-T/C/G mutation ratios at 256 motifs (NN-6mA-NN) on the single-stranded model DNA after mutation-prone primer extension. C) The mutation patterns (6mA to T, C, and G respectively) at 256 motifs (NN-6mA-NN) on the single-stranded model DNA. D) FTO demethylates over 99% methylated base of NN-6mA-NN model ssDNA in vitro and shows weaker activity on dsDNA. Data are mean ± s.e.m.; analyzed by two-tailed unpaired t-tests. The number of independently repeated reactions are shown in each plot. **** P <0.0001. E) Schematic diagram of DR-6mA-seq. Genomic DNA is sequentially denatured, mixed with spike-in probes, treated with FTO (or not), ligated to biotin-tagged adaptors, and extended by Bst 2.0 DNA polymerase or Q5 high-fidelity DNA polymerase. The synthesized DNA strand is enriched and amplified for next-generation sequencing. 6mA-to-T/C/G mutations were counted for defining 6mA sites and calculating 6mA methylation stoichiometry, using spike-in calibration curves. F) Mutation profiles (including 6mA to T, C, and G) of mutation-prone primer extension on the 6mA spike-in probes harboring 0%, 25%, 50%, 75% and 100% 6mA. Primer extension on FTO-treated probes and primer extension by Q5 high-fidelity DNA polymerase serve as two control groups and do not respond to 6mA fractions. See also Table S1 .

    Techniques Used: Mutagenesis, Methylation, In Vitro, Activity Assay, Two Tailed Test, Synthesized, Amplification, Next-Generation Sequencing

    A) The box plot of mutation ratio distribution at 6mA sites detected in E. coli gDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, i.e., Bst 2.0 extended DNA from untreated DNA (mutation group), Bst 2.0 extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). B) Venn diagram showing the overlapped 6mA sites among three biologically independent replicates, detected by DR-6mA-seq. C) Correlation analysis of methylation fractions in E. coli K-12 genome, indicating a high correlation between replicates of DR-6mA-seq. D) Histogram indicating the distribution of 6mA fractions at the modified sites in E. coli K-12 genome, revealed by DR-6mA-seq. E) Venn diagram showing the excellent overlap between DR-6mA-seq-detected 6mA sites and SMRT-detected 6mA sites in E. coli K-12 genome. F) Motif sequence logo and the list of consensus motifs containing 6mA sites in gDNA from E. coli K-12, uncovered by DR-6mA-seq. The frequency percentages of the top 5-base 6mA-containing motifs are shown. G) Correlation analysis of 6mA methylation fractions in E. coli K-12 genome, indicating a high correlation between 6mA sites detected by DR-6mA-seq and SMRT. See also Tables S2.
    Figure Legend Snippet: A) The box plot of mutation ratio distribution at 6mA sites detected in E. coli gDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, i.e., Bst 2.0 extended DNA from untreated DNA (mutation group), Bst 2.0 extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). B) Venn diagram showing the overlapped 6mA sites among three biologically independent replicates, detected by DR-6mA-seq. C) Correlation analysis of methylation fractions in E. coli K-12 genome, indicating a high correlation between replicates of DR-6mA-seq. D) Histogram indicating the distribution of 6mA fractions at the modified sites in E. coli K-12 genome, revealed by DR-6mA-seq. E) Venn diagram showing the excellent overlap between DR-6mA-seq-detected 6mA sites and SMRT-detected 6mA sites in E. coli K-12 genome. F) Motif sequence logo and the list of consensus motifs containing 6mA sites in gDNA from E. coli K-12, uncovered by DR-6mA-seq. The frequency percentages of the top 5-base 6mA-containing motifs are shown. G) Correlation analysis of 6mA methylation fractions in E. coli K-12 genome, indicating a high correlation between 6mA sites detected by DR-6mA-seq and SMRT. See also Tables S2.

    Techniques Used: Mutagenesis, Methylation, Modification, Sequencing

    A) The box plot of mutation ratio distribution at 6mA sites detected in HepG2 mtDNA, mouse brain mtDNA, and mouse liver mtDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 -extended DNA from untreated DNA (mutation group), Bst 2.0 -extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). n = 2, biologically independent replicates. B) The histogram of mtDNA 6mA site number distribution normalized to 6mA fraction bins, in HepG2 cells, mouse brain, and mouse liver. n = 2, biologically independent replicates. C) Distribution of 6mA sites along the human HepG2 mitochondrial genome. The dots on the lighter orange background are mtDNA 6mA sites on the forward and reverse strand of mtDNA of HepG2 replicate 1, while the dots on the darker orange background are mtDNA 6mA sites on the forward and reverse strand of mtDNA of HepG2 replicate 2, both revealed by DR-6mA-seq. The six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. The inner bright orange track represents overlapped mtDNA 6mA sites with 5-bp flanking windows. The inner purple track represents 6mA sites detected by ChIP-exo. D) Distribution of 6mA sites along the mouse mitochondrial genome. The dots on the purple and green background are mtDNA 6mA sites in two biological replicates of mouse brain and mouse liver, respectively, revealed by DR-6mA-seq, with six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. The inner red track indicates the regions of uniquely mapped reads in mouse mitochondria genome. E) Chromatogram of a representative injection of 6mA (0.001 pmol) and dG standard compound (800 pmol), using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼5.8 min. F) Chromatogram of a representative injection of 6mA (0.005 pmol) and dG standard compound (300 pmol) using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼5.8 min. G) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the first biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min. H) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the second biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min. I) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the third biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min.
    Figure Legend Snippet: A) The box plot of mutation ratio distribution at 6mA sites detected in HepG2 mtDNA, mouse brain mtDNA, and mouse liver mtDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 -extended DNA from untreated DNA (mutation group), Bst 2.0 -extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). n = 2, biologically independent replicates. B) The histogram of mtDNA 6mA site number distribution normalized to 6mA fraction bins, in HepG2 cells, mouse brain, and mouse liver. n = 2, biologically independent replicates. C) Distribution of 6mA sites along the human HepG2 mitochondrial genome. The dots on the lighter orange background are mtDNA 6mA sites on the forward and reverse strand of mtDNA of HepG2 replicate 1, while the dots on the darker orange background are mtDNA 6mA sites on the forward and reverse strand of mtDNA of HepG2 replicate 2, both revealed by DR-6mA-seq. The six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. The inner bright orange track represents overlapped mtDNA 6mA sites with 5-bp flanking windows. The inner purple track represents 6mA sites detected by ChIP-exo. D) Distribution of 6mA sites along the mouse mitochondrial genome. The dots on the purple and green background are mtDNA 6mA sites in two biological replicates of mouse brain and mouse liver, respectively, revealed by DR-6mA-seq, with six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. The inner red track indicates the regions of uniquely mapped reads in mouse mitochondria genome. E) Chromatogram of a representative injection of 6mA (0.001 pmol) and dG standard compound (800 pmol), using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼5.8 min. F) Chromatogram of a representative injection of 6mA (0.005 pmol) and dG standard compound (300 pmol) using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼5.8 min. G) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the first biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min. H) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the second biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min. I) Chromatogram of a representative injection of gDNA-depleted HEK293T mtDNA (the third biological replicate), using LC-MS/MS. The optimized LC protocol can distinguish the 6mA peak from the dT+Na + peak which appears at ∼4.2 min.

    Techniques Used: Mutagenesis, Methylation, Injection, Liquid Chromatography with Mass Spectroscopy

    A) Quantification of 6mA level in gDNA from human and mouse intact testis by LC-MS/MS, with E. coli gDNA as a positive control. Data are mean ± s.e.m.. B) Correlation analysis of 6mA methylation fractions in antibody-enriched mouse testis gDNA, indicating a high correlation between replicates of DR-6mA-seq. C) Venn diagram showing the overlapped 6mA sites in mouse testis gDNA, allowing and not allowing ± 1 kb window, detected by DR-6mA-seq between two biologically independent replicates. D) Motif sequence logo and the list of the consensus motifs containing 6mA sites in gDNA from mouse testis, uncovered by DR-6mA-seq. E) Chromosome-wide distributions of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. F) Genomic distributions of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. G) Genomic enrichment of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. See also , , and Tables S6.
    Figure Legend Snippet: A) Quantification of 6mA level in gDNA from human and mouse intact testis by LC-MS/MS, with E. coli gDNA as a positive control. Data are mean ± s.e.m.. B) Correlation analysis of 6mA methylation fractions in antibody-enriched mouse testis gDNA, indicating a high correlation between replicates of DR-6mA-seq. C) Venn diagram showing the overlapped 6mA sites in mouse testis gDNA, allowing and not allowing ± 1 kb window, detected by DR-6mA-seq between two biologically independent replicates. D) Motif sequence logo and the list of the consensus motifs containing 6mA sites in gDNA from mouse testis, uncovered by DR-6mA-seq. E) Chromosome-wide distributions of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. F) Genomic distributions of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. G) Genomic enrichment of gDNA 6mA sites in mouse testis, revealed by DR-6mA-seq. See also , , and Tables S6.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Positive Control, Methylation, Sequencing

    A) Chromatogram of a representative injection of 6mA standard compound, using LC-MS/MS. B) Chromatogram of a representative injection of the mock (nucleoside digestion mixture without adding any DNA) using LC-MS/MS. C) Chromatogram of a representative injection of digested human testis gDNA, using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼4.15 min. The optimized LC protocol distinguished the 6mA peak from the dT+Na + peak which appears at ∼3.00 min. D) Chromatogram of a representative injection of digested mouse testis gDNA, using LC-MS/MS. E) Chromatogram of a representative injection of E. coli K-12 gDNA, using LC-MS/MS. F) The box plot of mutation ratio distribution at 6mA sites detected in mouse testis gDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 -extended DNA from untreated DNA (mutation group), Bst 2.0 -extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control).
    Figure Legend Snippet: A) Chromatogram of a representative injection of 6mA standard compound, using LC-MS/MS. B) Chromatogram of a representative injection of the mock (nucleoside digestion mixture without adding any DNA) using LC-MS/MS. C) Chromatogram of a representative injection of digested human testis gDNA, using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼4.15 min. The optimized LC protocol distinguished the 6mA peak from the dT+Na + peak which appears at ∼3.00 min. D) Chromatogram of a representative injection of digested mouse testis gDNA, using LC-MS/MS. E) Chromatogram of a representative injection of E. coli K-12 gDNA, using LC-MS/MS. F) The box plot of mutation ratio distribution at 6mA sites detected in mouse testis gDNA, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 -extended DNA from untreated DNA (mutation group), Bst 2.0 -extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control).

    Techniques Used: Injection, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

    A) Chromatogram of a representative injection of 6mA standard compound, using LC-MS/MS. B) Chromatogram of a representative injection of the mock (nucleoside digestion mixture without adding any DNA) using LC-MS/MS. C) Chromatogram of a representative injection of digested gnotobiotic NIH/3T3 (ATCC) gDNA, using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼4.15 min. The optimized LC protocol distinguished the 6mA peak from the dT+Na + peak which appears at ∼3.00 min. D) Chromatogram of a representative injection of digested gnotobiotic B104-1-1 (ATCC) gDNA, using LC-MS/MS. E) Chromatogram of a representative injection of digested human liver gDNA, using LC-MS/MS. F) Chromatogram of a representative injection of digested human brain gDNA, using LC-MS/MS. G) Chromatogram of a representative injection of digested mouse E15.5 whole embryo gDNA, using LC-MS/MS. H) Chromatogram of a representative injection of E. coli K-12 gDNA, using LC-MS/MS.
    Figure Legend Snippet: A) Chromatogram of a representative injection of 6mA standard compound, using LC-MS/MS. B) Chromatogram of a representative injection of the mock (nucleoside digestion mixture without adding any DNA) using LC-MS/MS. C) Chromatogram of a representative injection of digested gnotobiotic NIH/3T3 (ATCC) gDNA, using LC-MS/MS. The 6mA quantification was calculated by the 6mA peak area, which displays a retention time of ∼4.15 min. The optimized LC protocol distinguished the 6mA peak from the dT+Na + peak which appears at ∼3.00 min. D) Chromatogram of a representative injection of digested gnotobiotic B104-1-1 (ATCC) gDNA, using LC-MS/MS. E) Chromatogram of a representative injection of digested human liver gDNA, using LC-MS/MS. F) Chromatogram of a representative injection of digested human brain gDNA, using LC-MS/MS. G) Chromatogram of a representative injection of digested mouse E15.5 whole embryo gDNA, using LC-MS/MS. H) Chromatogram of a representative injection of E. coli K-12 gDNA, using LC-MS/MS.

    Techniques Used: Injection, Liquid Chromatography with Mass Spectroscopy

    A) Quantification of 6mA level in gDNA from cultured NIH/3T3 and B104-1-1 cells, by LC-MS/MS, with E. coli gDNA as a positive control. Data are mean ± s.e.m. B) Correlation analysis of 6mA methylation fractions in antibody-enriched gDNA from NIH/3T3 and B104-1-1 cells, indicating a high correlation between replicates of DR-6mA-seq. C) Venn diagram showing the overlapped gDNA 6mA sites in NIH/3T3 and B104-1-1, allowing and not allowing ± 1 kb window, detected by DR-6mA-seq between two biologically independent replicates. D) Distributions of 6mA sites along the mouse genome. The dots on the blue and red background are gDNA 6mA sites in two biologically independent replicates of B104-1-1 and NIH/3T3 cells, respectively, revealed by DR-6mA-seq, with six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. E) Venn diagram showing the overlapped gDNA 6mA sites between NIH/3T3 and B104-1-1 cells, allowing ± 1 kb window. F) Genomic distributions of gDNA 6mA sites in NIH/3T3 and B104-1-1 cells, revealed by DR-6mA-seq. G) Genomic enrichment of gDNA 6mA sites identified in NIH/3T3 and B104-1-1 cells, revealed by DR-6mA-seq. Enrichment scores on intron and promoter are shown. See also - and Tables S7-S8.
    Figure Legend Snippet: A) Quantification of 6mA level in gDNA from cultured NIH/3T3 and B104-1-1 cells, by LC-MS/MS, with E. coli gDNA as a positive control. Data are mean ± s.e.m. B) Correlation analysis of 6mA methylation fractions in antibody-enriched gDNA from NIH/3T3 and B104-1-1 cells, indicating a high correlation between replicates of DR-6mA-seq. C) Venn diagram showing the overlapped gDNA 6mA sites in NIH/3T3 and B104-1-1, allowing and not allowing ± 1 kb window, detected by DR-6mA-seq between two biologically independent replicates. D) Distributions of 6mA sites along the mouse genome. The dots on the blue and red background are gDNA 6mA sites in two biologically independent replicates of B104-1-1 and NIH/3T3 cells, respectively, revealed by DR-6mA-seq, with six circled axes representing the methylation fractions at 0%, 20%, 40%, 60%, 80%, and 100%. E) Venn diagram showing the overlapped gDNA 6mA sites between NIH/3T3 and B104-1-1 cells, allowing ± 1 kb window. F) Genomic distributions of gDNA 6mA sites in NIH/3T3 and B104-1-1 cells, revealed by DR-6mA-seq. G) Genomic enrichment of gDNA 6mA sites identified in NIH/3T3 and B104-1-1 cells, revealed by DR-6mA-seq. Enrichment scores on intron and promoter are shown. See also - and Tables S7-S8.

    Techniques Used: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Positive Control, Methylation

    A) Genetic sex determination of NIH/3T3 and B104-1-1 cell lines by amplification of Rbm31x and Rbm31y by simplex PCR, with female and male mouse brains as controls. B) The box plot of mutation ratio distribution at 6mA sites detected in gDNA from NIH/3T3 (ATCC) and B104-1-1 (ATCC) cells, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 extended DNA from untreated DNA (mutation group), Bst 2.0 extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). C) Motif sequence logo and top 10 consensus motifs containing 6mA sites in gDNA from NIH/3T3 (ATCC) and B104-1-1 cells, uncovered by DR-6mA-seq. D) Chromosome-wide distributions of 6mA sites in gDNA from NIH/3T3. E) Chromosome-wide distributions of 6mA sites in gDNA from B104-1-1. F) Gene annotation shows the genomic distributions and enrichment of gDNA 6mA sites in NIH/3T3 cell lines, revealed by DR-6mA-seq. G) Gene annotation shows the genomic distributions and enrichment of gDNA 6mA sites identified in B104-1-1 cell lines, revealed by DR-6mA-seq. H) The enriched GO clusters of 6mA-modified genes in B104-1-1 cells. Genes with 6mA sites on exons, introns, and promoters are considered as 6mA-modified genes. I) Bright field photographs of NIH/3T3 and B104-1-1 cells.
    Figure Legend Snippet: A) Genetic sex determination of NIH/3T3 and B104-1-1 cell lines by amplification of Rbm31x and Rbm31y by simplex PCR, with female and male mouse brains as controls. B) The box plot of mutation ratio distribution at 6mA sites detected in gDNA from NIH/3T3 (ATCC) and B104-1-1 (ATCC) cells, revealed by DR-6mA-seq. The mutation ratios of three groups are shown, such as Bst 2.0 extended DNA from untreated DNA (mutation group), Bst 2.0 extended DNA from FTO-treated DNA (demethylation control), and Q5-extended DNA from untreated DNA (high-fidelity control). C) Motif sequence logo and top 10 consensus motifs containing 6mA sites in gDNA from NIH/3T3 (ATCC) and B104-1-1 cells, uncovered by DR-6mA-seq. D) Chromosome-wide distributions of 6mA sites in gDNA from NIH/3T3. E) Chromosome-wide distributions of 6mA sites in gDNA from B104-1-1. F) Gene annotation shows the genomic distributions and enrichment of gDNA 6mA sites in NIH/3T3 cell lines, revealed by DR-6mA-seq. G) Gene annotation shows the genomic distributions and enrichment of gDNA 6mA sites identified in B104-1-1 cell lines, revealed by DR-6mA-seq. H) The enriched GO clusters of 6mA-modified genes in B104-1-1 cells. Genes with 6mA sites on exons, introns, and promoters are considered as 6mA-modified genes. I) Bright field photographs of NIH/3T3 and B104-1-1 cells.

    Techniques Used: Amplification, Mutagenesis, Sequencing, Modification

    A) Two representative cell-line-specific gDNA 6mA clusters located at Abcc5 and Erich6 in B104-1-1 cells. B) RT-qPCR quantification of Bmpr1b expression level in NIH/3T3 and B104-1-1 cells, normalized to Actb . Data are mean ± s.e.m.; analyzed by two-tailed unpaired t-tests. The number of independently repeated reactions is shown in each plot. **** P <0.0001. C) The NIH/3T3 gDNA 6mA site number overlapped with ATAC-seq peaks from wild-type NIH/3T3 cells (GSE119781), by setting a sliding window of 500 bp, 1,000 bp, or 2,000 bp centered at the 6mA site revealed by DR-6mA-seq. D) The B104-1-1 gDNA 6mA site number overlapped with ATAC-seq peaks from glioblastoma tumor induced by implanted GL261 cells (GSE206551), by setting a sliding window of 500 bp, 1,000 bp, or 2,000 bp centered at the 6mA site revealed by DR-6mA-seq. E) The 2-D plot of NIH/3T3 gDNA 6mA methylation fraction versus the distance to ATAC-seq peak center (GSE119781), most 6mA sites distribute within ±1,000 bp range around ATAC-seq peak center. F) The 2-D plot of B104-1-1 gDNA 6mA methylation fraction versus the distance to ATAC-seq peak center (GSE206551), most 6mA sites distribute within ±1,500 bp range around ATAC-seq peak center.
    Figure Legend Snippet: A) Two representative cell-line-specific gDNA 6mA clusters located at Abcc5 and Erich6 in B104-1-1 cells. B) RT-qPCR quantification of Bmpr1b expression level in NIH/3T3 and B104-1-1 cells, normalized to Actb . Data are mean ± s.e.m.; analyzed by two-tailed unpaired t-tests. The number of independently repeated reactions is shown in each plot. **** P <0.0001. C) The NIH/3T3 gDNA 6mA site number overlapped with ATAC-seq peaks from wild-type NIH/3T3 cells (GSE119781), by setting a sliding window of 500 bp, 1,000 bp, or 2,000 bp centered at the 6mA site revealed by DR-6mA-seq. D) The B104-1-1 gDNA 6mA site number overlapped with ATAC-seq peaks from glioblastoma tumor induced by implanted GL261 cells (GSE206551), by setting a sliding window of 500 bp, 1,000 bp, or 2,000 bp centered at the 6mA site revealed by DR-6mA-seq. E) The 2-D plot of NIH/3T3 gDNA 6mA methylation fraction versus the distance to ATAC-seq peak center (GSE119781), most 6mA sites distribute within ±1,000 bp range around ATAC-seq peak center. F) The 2-D plot of B104-1-1 gDNA 6mA methylation fraction versus the distance to ATAC-seq peak center (GSE206551), most 6mA sites distribute within ±1,500 bp range around ATAC-seq peak center.

    Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test, Methylation

    A) A flowchart of amplicon assay for gDNA 6mA validation, revealing 6mA fraction by adjusted misincorporation ratios. B) The actual misincorporation ratios obtained in amplicon assay versus the misincorporation calculated from DR-6mA-seq data. C) The 6mA methylation fractions by amplicon assay versus 6mA fractions calculated from DR-6mA-seq data. gDNA 6mA sites showing >70% estimated modification in amplicon assay were marked by red arrows; other gDNA 6mA sites showing above 10% modification fraction were labeled by green arrows. For B) and C), 16 gDNA 6mA sites were investigated, with 6 from NIH/3T3 cells and 10 from B104-1-1 cells. D) A representative 6mA cluster located at Bmpr1b , with the arrow marking the 6mA site of the highest modification fraction identified in C). See also - .
    Figure Legend Snippet: A) A flowchart of amplicon assay for gDNA 6mA validation, revealing 6mA fraction by adjusted misincorporation ratios. B) The actual misincorporation ratios obtained in amplicon assay versus the misincorporation calculated from DR-6mA-seq data. C) The 6mA methylation fractions by amplicon assay versus 6mA fractions calculated from DR-6mA-seq data. gDNA 6mA sites showing >70% estimated modification in amplicon assay were marked by red arrows; other gDNA 6mA sites showing above 10% modification fraction were labeled by green arrows. For B) and C), 16 gDNA 6mA sites were investigated, with 6 from NIH/3T3 cells and 10 from B104-1-1 cells. D) A representative 6mA cluster located at Bmpr1b , with the arrow marking the 6mA site of the highest modification fraction identified in C). See also - .

    Techniques Used: Amplification, Methylation, Modification, Labeling

    monarch genomic dna purification kit  (New England Biolabs)


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    Gene copy number of insertion and tandem duplication lines. Copy number in homozygous male flies was measured in duplex digital PCR assays, with count of focal gene ( w or Adh ) normalized to count of autosomal control gene RpL32 . Each point is a measurement of a separate single-fly <t>genomic</t> <t>DNA</t> preparation, n=3 per genotype. The w assay also detects the w − allele from the endogenous X-linked locus, which is expected to contribute 0.5 copies in these hemizygous males.
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    1) Product Images from "Demonstration of in vivo engineered tandem duplications of varying sizes using CRISPR and recombinases in Drosophila melanogaster"

    Article Title: Demonstration of in vivo engineered tandem duplications of varying sizes using CRISPR and recombinases in Drosophila melanogaster

    Journal: bioRxiv

    doi: 10.1101/2023.01.08.523181

    Gene copy number of insertion and tandem duplication lines. Copy number in homozygous male flies was measured in duplex digital PCR assays, with count of focal gene ( w or Adh ) normalized to count of autosomal control gene RpL32 . Each point is a measurement of a separate single-fly genomic DNA preparation, n=3 per genotype. The w assay also detects the w − allele from the endogenous X-linked locus, which is expected to contribute 0.5 copies in these hemizygous males.
    Figure Legend Snippet: Gene copy number of insertion and tandem duplication lines. Copy number in homozygous male flies was measured in duplex digital PCR assays, with count of focal gene ( w or Adh ) normalized to count of autosomal control gene RpL32 . Each point is a measurement of a separate single-fly genomic DNA preparation, n=3 per genotype. The w assay also detects the w − allele from the endogenous X-linked locus, which is expected to contribute 0.5 copies in these hemizygous males.

    Techniques Used: Digital PCR

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    New England Biolabs monarch genomic dna purification kit
    ( A ) Principle of the bs-ATLAS-seq method. The internal L1 promoter region (∼ 900 bp) is illustrated (top). Transcription start sites for the sense (SP) and antisense (ASP) promoters are represented as broken arrows and overlap with L1 5’UTR. bs-ATLAS-seq interrogates the first 15 CpG sites of the L1 promoter, shown as vertical bars in the magnified view (bottom). The L1-specific primer used to amplify L1 junctions is shown as a green arrow. <t>Genomic</t> <t>DNA</t> is fragmented by sonication and ligated to a single-stranded methylated linker. Linker-ligated DNA is then treated with bisulfite and L1-containing fragments are specifically amplified by suppression PCR. The suppression PCR step is designed to enrich for the L1HS family. Finally, asymmetric paired-end sequencing provides the genomic location as well as the methylation levels of each L1 locus. R1 and R2 refers to reads #1 and #2, respectively. Note that 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are both protected from bisulfite-induced deamination. Thus bs-ATLAS-seq cannot discriminate between these two DNA modifications. ( B, C ) Genome browser view of bs-ATLAS-seq results in the breast cancer cell line MCF-7 for a reference (B) and a non-reference (C) L1HS element in the TTC28 and NEDD4 genes, respectively. In the track showing the percentage of methylation, called CpG are indicated by a vertical gray bar, and the percentage of methylation as an overlapping black bar. In the ‘coverage’ and ‘reads’ tracks, vertical colored bars correspond to non-methylated CpG (blue) and methylated CpG (mCG, red). Since bisulfite-sequencing-based methods cannot discriminate between hydroxymethylated CpG (hmCG) and methylated-CpG (mCG), methylation status is indicated as mCG + hmCG. (C) For non-reference L1HS, only the genomic flank covered by read #1 (R1 ; bottom left) is visible in the genome browser view. Soft-clipped reads supporting the 5’ L1 junction (split reads) are framed in pink. The proportion of mCG at each site and the frequency of the most common methylation patterns deduced from read 2 (R2 ; bottom right) are indicated on the charts (right). Positions of CpGs are related to L1HS consensus sequence (see Methods). (D) Number of full-length L1HS elements detected in the different cell lines by bs-ATLAS-seq and subfamilies. Pre-Ta, Ta0 and Ta1 represent different lineages of the L1HS family, from the oldest to the youngest, and were deduced from diagnostic nucleotides in L1 internal sequence (Boissinot et al., 2000) and thus could only be obtained for reference insertions as bs-ATLAS-seq provides only limited information on L1 internal sequence. (E) Fraction and count of reference L1 elements detected by bs-ATLAS-seq for each L1 family. Bars represent the average number of reference L1 elements detected by bs-ATLAS-seq (dark green, mean ± s.d., n=12 cell lines), as compared to the total number of these elements in the reference genome (light green). The ratio of detected/total elements is indicated as a percentage on the right of each bar. Note that any given sample only contains a subset of reference L1HS due to insertional polymorphisms in the human population. See also and Table S1 .
    Monarch Genomic Dna Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Resolving the heterogeneity of L1 DNA methylation reveals the epigenetic and transcriptional interplay between L1s and their integration sites"

    Article Title: Resolving the heterogeneity of L1 DNA methylation reveals the epigenetic and transcriptional interplay between L1s and their integration sites

    Journal: bioRxiv

    doi: 10.1101/2023.01.03.522582

    ( A ) Principle of the bs-ATLAS-seq method. The internal L1 promoter region (∼ 900 bp) is illustrated (top). Transcription start sites for the sense (SP) and antisense (ASP) promoters are represented as broken arrows and overlap with L1 5’UTR. bs-ATLAS-seq interrogates the first 15 CpG sites of the L1 promoter, shown as vertical bars in the magnified view (bottom). The L1-specific primer used to amplify L1 junctions is shown as a green arrow. Genomic DNA is fragmented by sonication and ligated to a single-stranded methylated linker. Linker-ligated DNA is then treated with bisulfite and L1-containing fragments are specifically amplified by suppression PCR. The suppression PCR step is designed to enrich for the L1HS family. Finally, asymmetric paired-end sequencing provides the genomic location as well as the methylation levels of each L1 locus. R1 and R2 refers to reads #1 and #2, respectively. Note that 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are both protected from bisulfite-induced deamination. Thus bs-ATLAS-seq cannot discriminate between these two DNA modifications. ( B, C ) Genome browser view of bs-ATLAS-seq results in the breast cancer cell line MCF-7 for a reference (B) and a non-reference (C) L1HS element in the TTC28 and NEDD4 genes, respectively. In the track showing the percentage of methylation, called CpG are indicated by a vertical gray bar, and the percentage of methylation as an overlapping black bar. In the ‘coverage’ and ‘reads’ tracks, vertical colored bars correspond to non-methylated CpG (blue) and methylated CpG (mCG, red). Since bisulfite-sequencing-based methods cannot discriminate between hydroxymethylated CpG (hmCG) and methylated-CpG (mCG), methylation status is indicated as mCG + hmCG. (C) For non-reference L1HS, only the genomic flank covered by read #1 (R1 ; bottom left) is visible in the genome browser view. Soft-clipped reads supporting the 5’ L1 junction (split reads) are framed in pink. The proportion of mCG at each site and the frequency of the most common methylation patterns deduced from read 2 (R2 ; bottom right) are indicated on the charts (right). Positions of CpGs are related to L1HS consensus sequence (see Methods). (D) Number of full-length L1HS elements detected in the different cell lines by bs-ATLAS-seq and subfamilies. Pre-Ta, Ta0 and Ta1 represent different lineages of the L1HS family, from the oldest to the youngest, and were deduced from diagnostic nucleotides in L1 internal sequence (Boissinot et al., 2000) and thus could only be obtained for reference insertions as bs-ATLAS-seq provides only limited information on L1 internal sequence. (E) Fraction and count of reference L1 elements detected by bs-ATLAS-seq for each L1 family. Bars represent the average number of reference L1 elements detected by bs-ATLAS-seq (dark green, mean ± s.d., n=12 cell lines), as compared to the total number of these elements in the reference genome (light green). The ratio of detected/total elements is indicated as a percentage on the right of each bar. Note that any given sample only contains a subset of reference L1HS due to insertional polymorphisms in the human population. See also and Table S1 .
    Figure Legend Snippet: ( A ) Principle of the bs-ATLAS-seq method. The internal L1 promoter region (∼ 900 bp) is illustrated (top). Transcription start sites for the sense (SP) and antisense (ASP) promoters are represented as broken arrows and overlap with L1 5’UTR. bs-ATLAS-seq interrogates the first 15 CpG sites of the L1 promoter, shown as vertical bars in the magnified view (bottom). The L1-specific primer used to amplify L1 junctions is shown as a green arrow. Genomic DNA is fragmented by sonication and ligated to a single-stranded methylated linker. Linker-ligated DNA is then treated with bisulfite and L1-containing fragments are specifically amplified by suppression PCR. The suppression PCR step is designed to enrich for the L1HS family. Finally, asymmetric paired-end sequencing provides the genomic location as well as the methylation levels of each L1 locus. R1 and R2 refers to reads #1 and #2, respectively. Note that 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are both protected from bisulfite-induced deamination. Thus bs-ATLAS-seq cannot discriminate between these two DNA modifications. ( B, C ) Genome browser view of bs-ATLAS-seq results in the breast cancer cell line MCF-7 for a reference (B) and a non-reference (C) L1HS element in the TTC28 and NEDD4 genes, respectively. In the track showing the percentage of methylation, called CpG are indicated by a vertical gray bar, and the percentage of methylation as an overlapping black bar. In the ‘coverage’ and ‘reads’ tracks, vertical colored bars correspond to non-methylated CpG (blue) and methylated CpG (mCG, red). Since bisulfite-sequencing-based methods cannot discriminate between hydroxymethylated CpG (hmCG) and methylated-CpG (mCG), methylation status is indicated as mCG + hmCG. (C) For non-reference L1HS, only the genomic flank covered by read #1 (R1 ; bottom left) is visible in the genome browser view. Soft-clipped reads supporting the 5’ L1 junction (split reads) are framed in pink. The proportion of mCG at each site and the frequency of the most common methylation patterns deduced from read 2 (R2 ; bottom right) are indicated on the charts (right). Positions of CpGs are related to L1HS consensus sequence (see Methods). (D) Number of full-length L1HS elements detected in the different cell lines by bs-ATLAS-seq and subfamilies. Pre-Ta, Ta0 and Ta1 represent different lineages of the L1HS family, from the oldest to the youngest, and were deduced from diagnostic nucleotides in L1 internal sequence (Boissinot et al., 2000) and thus could only be obtained for reference insertions as bs-ATLAS-seq provides only limited information on L1 internal sequence. (E) Fraction and count of reference L1 elements detected by bs-ATLAS-seq for each L1 family. Bars represent the average number of reference L1 elements detected by bs-ATLAS-seq (dark green, mean ± s.d., n=12 cell lines), as compared to the total number of these elements in the reference genome (light green). The ratio of detected/total elements is indicated as a percentage on the right of each bar. Note that any given sample only contains a subset of reference L1HS due to insertional polymorphisms in the human population. See also and Table S1 .

    Techniques Used: Sonication, Methylation, Amplification, Sequencing, Methylation Sequencing, Diagnostic Assay

    ( A , B ) Genome browser view of bs-ATLAS-seq results in BJ foreskin fibroblasts at the reference L1HS-TTC28 locus ( A ) and at the non-reference L1HS-NEDD4 locus ( B ). In the track showing the percentage of methylation, called CpG are indicated by a vertical gray bar, and the percentage of methylation as an overlapping black bar. In the ‘coverage’ and ‘reads’ tracks, vertical colored bars correspond to non-methylated CpG (blue) and methylated CpG (mCG, red). For non-reference L1HS, only the genomic flank covered by read #1 (R1 ; bottom left) is visible in the genome browser view. Soft-clipped reads supporting the 5’ L1 junction (split reads) are framed in pink. The proportion of mCG at each site and the frequency of the most common methylation patterns deduced from read 2 (R2 ; bottom right) are indicated on the charts (right). Positions of CpGs are related to L1HS consensus sequence (see Methods). ( C ) Sensitivity analysis. Computational down-sampling of high depth bs-ATLAS-seq sequencing data (MCF7) shows that L1 recovery reaches a plateau above 10 million of total read pairs. Thus, all samples were subsequently sequenced to a depth greater than 10 million of total reads. ( D, E ) Reproducibility of bs-ATLAS-seq. Two independent libraries of MCF-7 (from two subsequent MCF-7 passages) and sequencing runs are compared with respect to L1 elements of all families ( E ) or to L1HS elements only ( F ). Replicate 1 (MCF7, Rep1) was down sampled to the sequencing depth of replicate 2 (MCF7, Rep2) for comparison purpose. Left panels, correlation of methylation levels for shared detected L1 loci. Right panels, Venn diagram showing the overlap of detected L1 loci between the two libraries. (F) Statistics of bs-ATLAS-seq for the 12-cell line panel. See also Table S1 . (G) PCR validation of unknown non-reference insertions. PCR was done with genomic DNA of the indicated cell lines (+) or with water as non-template control (-). The insertion in chr10 is pericentromeric and embedded in other repeats. Therefore, only primers to amplify the 5’ junction could be designed. Ct, unrelated locus used as PCR control. (H) DNA methylation level of selected L1 elements was profiled using an antibody-based enrichment of DNA methylation (MeDIP) and compared with bs-ATLAS-seq data. The DNA methylation level of MeDIP data is expressed as log2 of the percentage of immunoprecipitation (log2 %IP). See also Table S2 . (I) Comparison of bs-ATLAS-seq with PCR-free targeted sequencing and methylation calling by Oxford Nanopore sequencing (ONT-seq) for the L1 region common to both methods (1-207) or the full CpG island (1-500). See also Table S4 . For all correlation analysis, r and p represent Pearson correlation coefficient and p-value, respectively.
    Figure Legend Snippet: ( A , B ) Genome browser view of bs-ATLAS-seq results in BJ foreskin fibroblasts at the reference L1HS-TTC28 locus ( A ) and at the non-reference L1HS-NEDD4 locus ( B ). In the track showing the percentage of methylation, called CpG are indicated by a vertical gray bar, and the percentage of methylation as an overlapping black bar. In the ‘coverage’ and ‘reads’ tracks, vertical colored bars correspond to non-methylated CpG (blue) and methylated CpG (mCG, red). For non-reference L1HS, only the genomic flank covered by read #1 (R1 ; bottom left) is visible in the genome browser view. Soft-clipped reads supporting the 5’ L1 junction (split reads) are framed in pink. The proportion of mCG at each site and the frequency of the most common methylation patterns deduced from read 2 (R2 ; bottom right) are indicated on the charts (right). Positions of CpGs are related to L1HS consensus sequence (see Methods). ( C ) Sensitivity analysis. Computational down-sampling of high depth bs-ATLAS-seq sequencing data (MCF7) shows that L1 recovery reaches a plateau above 10 million of total read pairs. Thus, all samples were subsequently sequenced to a depth greater than 10 million of total reads. ( D, E ) Reproducibility of bs-ATLAS-seq. Two independent libraries of MCF-7 (from two subsequent MCF-7 passages) and sequencing runs are compared with respect to L1 elements of all families ( E ) or to L1HS elements only ( F ). Replicate 1 (MCF7, Rep1) was down sampled to the sequencing depth of replicate 2 (MCF7, Rep2) for comparison purpose. Left panels, correlation of methylation levels for shared detected L1 loci. Right panels, Venn diagram showing the overlap of detected L1 loci between the two libraries. (F) Statistics of bs-ATLAS-seq for the 12-cell line panel. See also Table S1 . (G) PCR validation of unknown non-reference insertions. PCR was done with genomic DNA of the indicated cell lines (+) or with water as non-template control (-). The insertion in chr10 is pericentromeric and embedded in other repeats. Therefore, only primers to amplify the 5’ junction could be designed. Ct, unrelated locus used as PCR control. (H) DNA methylation level of selected L1 elements was profiled using an antibody-based enrichment of DNA methylation (MeDIP) and compared with bs-ATLAS-seq data. The DNA methylation level of MeDIP data is expressed as log2 of the percentage of immunoprecipitation (log2 %IP). See also Table S2 . (I) Comparison of bs-ATLAS-seq with PCR-free targeted sequencing and methylation calling by Oxford Nanopore sequencing (ONT-seq) for the L1 region common to both methods (1-207) or the full CpG island (1-500). See also Table S4 . For all correlation analysis, r and p represent Pearson correlation coefficient and p-value, respectively.

    Techniques Used: Methylation, Sequencing, Sampling, DNA Methylation Assay, Methylated DNA Immunoprecipitation, Immunoprecipitation, Nanopore Sequencing

    (A) Scheme summarizing the strategy to genotype and assess DNA methylation profiles of loci containing polymorphic L1HS elements by Cas9-guided nanopore sequencing. Single-guide RNAs (sgRNAs) designed to bind ∼ 1 kb downstream of each L1HS insertion (green solid arrows), were synthesized in vitro as a pool and assembled with recombinant Cas9. Cleavage of genomic DNA with the pool of Cas9 RNPs allows the subsequent ligation of sequencing adapters downstream of L1s and targeted nanopore sequencing of the selected loci (see Methods). As an example, a genome browser screenshot is shown for the insertion chr3:85527420-85527422:+:L1HS:NONREF in 2102Ep cells (top: filled allele, bottom: empty allele). The targeted L1HS (green), as well as methylated (red) and unmethylated (blue) CpG are indicated. Only the first 10 reads are shown for each allele, and the 3’ end of the reads (on the left) have been truncated for layout. (B) Theoretical methylation profiles at heterozygous L1 insertions (green solid arrow). The influence of L1 methylation on the surrounding genomic region can only be detected in situations where the methylation state of L1 differs from that of the empty locus (assumed to represent the pre-integration state). For these informative scenarios, the DNA methylation level of the region upstream of L1 is then analyzed and compared to the empty locus. (C) Characteristics of the loci profiled by nanopore sequencing. The y-axis represents the number of loci characterized in the 4 cell lines as filled, heterozygous and with CpG sites upstream of L1 (∼ 300 bp). Among them, 37 loci were considered as informative: 23 with a profile consistent with L1-induced epivariation, 10 showing no evidence of L1-induced epivariation, and 4 being inconclusive (see Methods). (D) L1-driven hypomethylation of the proximal upstream genomic region. Left, average DNA methylation levels in 100 bp-bins at loci with an upstream slopping shore (n=14, mean ± s.d.). Middle and right, examples of L1-induced upstream flank hypomethylation. (E) L1-driven methylation of the proximal flanking genomic region. Left, average DNA methylation levels in 100 bp-bins at loci with DNA methylation spreading from L1 to the external flanks (n=9, mean ± s.d.). Middle and right, examples of L1-induced flanking sequence DNA methylation. For ( D ) and ( E ), the empty (white squares) and filled (green circles) alleles are overlaid. Blue and red arrows denote hypo- and hyper-methylation relative to the empty locus, respectively. The x-axis represents the relative distance to L1 5’ end (bp) and the y-axis the percentage of DNA methylation. See also and Table S4 .
    Figure Legend Snippet: (A) Scheme summarizing the strategy to genotype and assess DNA methylation profiles of loci containing polymorphic L1HS elements by Cas9-guided nanopore sequencing. Single-guide RNAs (sgRNAs) designed to bind ∼ 1 kb downstream of each L1HS insertion (green solid arrows), were synthesized in vitro as a pool and assembled with recombinant Cas9. Cleavage of genomic DNA with the pool of Cas9 RNPs allows the subsequent ligation of sequencing adapters downstream of L1s and targeted nanopore sequencing of the selected loci (see Methods). As an example, a genome browser screenshot is shown for the insertion chr3:85527420-85527422:+:L1HS:NONREF in 2102Ep cells (top: filled allele, bottom: empty allele). The targeted L1HS (green), as well as methylated (red) and unmethylated (blue) CpG are indicated. Only the first 10 reads are shown for each allele, and the 3’ end of the reads (on the left) have been truncated for layout. (B) Theoretical methylation profiles at heterozygous L1 insertions (green solid arrow). The influence of L1 methylation on the surrounding genomic region can only be detected in situations where the methylation state of L1 differs from that of the empty locus (assumed to represent the pre-integration state). For these informative scenarios, the DNA methylation level of the region upstream of L1 is then analyzed and compared to the empty locus. (C) Characteristics of the loci profiled by nanopore sequencing. The y-axis represents the number of loci characterized in the 4 cell lines as filled, heterozygous and with CpG sites upstream of L1 (∼ 300 bp). Among them, 37 loci were considered as informative: 23 with a profile consistent with L1-induced epivariation, 10 showing no evidence of L1-induced epivariation, and 4 being inconclusive (see Methods). (D) L1-driven hypomethylation of the proximal upstream genomic region. Left, average DNA methylation levels in 100 bp-bins at loci with an upstream slopping shore (n=14, mean ± s.d.). Middle and right, examples of L1-induced upstream flank hypomethylation. (E) L1-driven methylation of the proximal flanking genomic region. Left, average DNA methylation levels in 100 bp-bins at loci with DNA methylation spreading from L1 to the external flanks (n=9, mean ± s.d.). Middle and right, examples of L1-induced flanking sequence DNA methylation. For ( D ) and ( E ), the empty (white squares) and filled (green circles) alleles are overlaid. Blue and red arrows denote hypo- and hyper-methylation relative to the empty locus, respectively. The x-axis represents the relative distance to L1 5’ end (bp) and the y-axis the percentage of DNA methylation. See also and Table S4 .

    Techniques Used: DNA Methylation Assay, Nanopore Sequencing, Synthesized, In Vitro, Recombinant, Ligation, Sequencing, Methylation

    (A) ORF1p immunoblot of L1 RNP preparations from various cell types. Quantities of RNP loaded are indicated at the bottom of the blots. S6 immunoblot was used as a loading control. Molecular weight marker (kDa) is indicated on the left. Panel taken from (Philippe et al., 2016). (B) Comparison of expressed L1 elements detected by the 3’ readthrough approach and L1EM. Left, barplots indicating the absolute number of L1HS elements with low (mCG ≤ 25%), medium (25% < mCG < 75%) or high (mCG ≥ 75%) methylation according to bs-ATLAS-seq data, and being detected as unexpressed (white) or expressed (light green) by L1EM. Right, Venn diagram showing the overlap of L1HS detected as expressed by the two methods. (C) Metaplot of L1 DNA methylation profiles (bs-ATLAS-seq) at the L1HS promoter and upstream flanking region (300 bp) in HCT-116 treated with 5-aza-2-deoxycytidine (two replicates, AZA #1 and AZA#2; dark green) or with DMSO as negative control (two replicates: DMSO #1 and DMSO #2; light green). (D) Fraction of fully unmethylated reads in 5-aza-or DMSO-treated HCT-116 cells. Boxplots represent the median and interquartile range (IQR) ± 1.5 * IQR (whiskers). Outliers beyond the end of the whiskers are plotted individually. See (C) for legend. (E) Differential expression of transposable element (TE) families between 5-aza-or DMSO-treated HCT-116 cells measured by poly(A) + RNA-seq using TEtranscripts . In the MA-plot, each data point represents an aggregated TE family. TE families found significantly up-or down-regulated upon 5-aza treatment are colored in purple and green, respectively, and data points corresponding to the L1HS to L1PA8 families are labelled (of which all but L1PA8 are upregulated). (F) Heatmaps showing the average difference of L1HS methylation (ΔmCG, bs-ATLAS-seq, 2 replicates) between HCT-116 cells treated by DMSO and 5-aza (AZA), as well as the expression levels of each L1HS in RNA-seq replicates (L1 3’ readthrough, see legend of Figure 6A). Heatmaps were sorted by decreasing L1 expression (average of the 3 replicates). (G) L1HS expression vs methylation in 5-aza-treated HCT-116 cells. L1 methylation is defined here as the mean fraction of fully unmethylated reads per L1HS locus and per condition (AZA or DMSO) and expression is estimated through L1 3’ readthrough as described in the legend of Figure 6A. Each point represents an L1HS locus and a replicate and was colored in light (DMSO) or dark (AZA) green. (H) Comparison of the expression and methylation levels of an intronic L1HS insertion located in the CASC21 gene, across cell types and conditions. This element is only expressed upon 5-aza treatment, but not in other cell types with similar or even completely abolished methylation (MCF-7, K-562, 2102Ep or HEK-293T). (I) Genome browser view of the L1HS-CASC21 locus treated (AZA) or not (DMSO) by 5-aza, integrating L1 methylation (bs-ATLAS-seq) and expression (poly(A)+ RNA-seq).
    Figure Legend Snippet: (A) ORF1p immunoblot of L1 RNP preparations from various cell types. Quantities of RNP loaded are indicated at the bottom of the blots. S6 immunoblot was used as a loading control. Molecular weight marker (kDa) is indicated on the left. Panel taken from (Philippe et al., 2016). (B) Comparison of expressed L1 elements detected by the 3’ readthrough approach and L1EM. Left, barplots indicating the absolute number of L1HS elements with low (mCG ≤ 25%), medium (25% < mCG < 75%) or high (mCG ≥ 75%) methylation according to bs-ATLAS-seq data, and being detected as unexpressed (white) or expressed (light green) by L1EM. Right, Venn diagram showing the overlap of L1HS detected as expressed by the two methods. (C) Metaplot of L1 DNA methylation profiles (bs-ATLAS-seq) at the L1HS promoter and upstream flanking region (300 bp) in HCT-116 treated with 5-aza-2-deoxycytidine (two replicates, AZA #1 and AZA#2; dark green) or with DMSO as negative control (two replicates: DMSO #1 and DMSO #2; light green). (D) Fraction of fully unmethylated reads in 5-aza-or DMSO-treated HCT-116 cells. Boxplots represent the median and interquartile range (IQR) ± 1.5 * IQR (whiskers). Outliers beyond the end of the whiskers are plotted individually. See (C) for legend. (E) Differential expression of transposable element (TE) families between 5-aza-or DMSO-treated HCT-116 cells measured by poly(A) + RNA-seq using TEtranscripts . In the MA-plot, each data point represents an aggregated TE family. TE families found significantly up-or down-regulated upon 5-aza treatment are colored in purple and green, respectively, and data points corresponding to the L1HS to L1PA8 families are labelled (of which all but L1PA8 are upregulated). (F) Heatmaps showing the average difference of L1HS methylation (ΔmCG, bs-ATLAS-seq, 2 replicates) between HCT-116 cells treated by DMSO and 5-aza (AZA), as well as the expression levels of each L1HS in RNA-seq replicates (L1 3’ readthrough, see legend of Figure 6A). Heatmaps were sorted by decreasing L1 expression (average of the 3 replicates). (G) L1HS expression vs methylation in 5-aza-treated HCT-116 cells. L1 methylation is defined here as the mean fraction of fully unmethylated reads per L1HS locus and per condition (AZA or DMSO) and expression is estimated through L1 3’ readthrough as described in the legend of Figure 6A. Each point represents an L1HS locus and a replicate and was colored in light (DMSO) or dark (AZA) green. (H) Comparison of the expression and methylation levels of an intronic L1HS insertion located in the CASC21 gene, across cell types and conditions. This element is only expressed upon 5-aza treatment, but not in other cell types with similar or even completely abolished methylation (MCF-7, K-562, 2102Ep or HEK-293T). (I) Genome browser view of the L1HS-CASC21 locus treated (AZA) or not (DMSO) by 5-aza, integrating L1 methylation (bs-ATLAS-seq) and expression (poly(A)+ RNA-seq).

    Techniques Used: Western Blot, Molecular Weight, Marker, Methylation, DNA Methylation Assay, Negative Control, Expressing, RNA Sequencing Assay

    monarch genomic dna purification kit  (New England Biolabs)


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    New England Biolabs monarch genomic dna purification kit
    Monarch Genomic Dna Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs monarch genomic dna purification kit
    Early uptake of adenovirus 5 as control and 13 species D adenovirus into CHO cells. The cells were infected with 1000 vpc of each virus with or without 10 µg/mL FX. At 3 <t>hpi,</t> <t>gDNA</t> was isolated and duplex qPCR detecting GAPDH as housekeeping gene and neomycin contained in the Ad genome was performed. The infectious particles in the different cell lines are displayed in blue (CHO-K1 cells), grey (CHO-606 cells) and orange (CHO-745 cells). ( A ). As proof of concept, the CHO cells were infected with 1000 vpc of adenovirus 5 with and without FX. The early uptake of viral particles was 3-fold higher with FX than without in CHO-K1 cells. The number of infectious particles stayed the same in CHO-606 cells with and without FX, and in the CHO-745 cells with no heparan chains, the uptake of particles was very low under both conditions. N = 2 biological replicates. ( B ). The CHO cell lines were infected with 1000 vpc of each species D adenovirus. After 3 hpi no effect of FX was seen either in wild type HSPG CHO cells or in the HSPG depleted cells. Mean ± SEM of n = 3 biological replicates is shown.
    Monarch Genomic Dna Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Influence of Heparan Sulfate Proteoglycans and Factor X on species D Human Adenovirus Uptake and Transduction"

    Article Title: Influence of Heparan Sulfate Proteoglycans and Factor X on species D Human Adenovirus Uptake and Transduction

    Journal: Viruses

    doi: 10.3390/v15010055

    Early uptake of adenovirus 5 as control and 13 species D adenovirus into CHO cells. The cells were infected with 1000 vpc of each virus with or without 10 µg/mL FX. At 3 hpi, gDNA was isolated and duplex qPCR detecting GAPDH as housekeeping gene and neomycin contained in the Ad genome was performed. The infectious particles in the different cell lines are displayed in blue (CHO-K1 cells), grey (CHO-606 cells) and orange (CHO-745 cells). ( A ). As proof of concept, the CHO cells were infected with 1000 vpc of adenovirus 5 with and without FX. The early uptake of viral particles was 3-fold higher with FX than without in CHO-K1 cells. The number of infectious particles stayed the same in CHO-606 cells with and without FX, and in the CHO-745 cells with no heparan chains, the uptake of particles was very low under both conditions. N = 2 biological replicates. ( B ). The CHO cell lines were infected with 1000 vpc of each species D adenovirus. After 3 hpi no effect of FX was seen either in wild type HSPG CHO cells or in the HSPG depleted cells. Mean ± SEM of n = 3 biological replicates is shown.
    Figure Legend Snippet: Early uptake of adenovirus 5 as control and 13 species D adenovirus into CHO cells. The cells were infected with 1000 vpc of each virus with or without 10 µg/mL FX. At 3 hpi, gDNA was isolated and duplex qPCR detecting GAPDH as housekeeping gene and neomycin contained in the Ad genome was performed. The infectious particles in the different cell lines are displayed in blue (CHO-K1 cells), grey (CHO-606 cells) and orange (CHO-745 cells). ( A ). As proof of concept, the CHO cells were infected with 1000 vpc of adenovirus 5 with and without FX. The early uptake of viral particles was 3-fold higher with FX than without in CHO-K1 cells. The number of infectious particles stayed the same in CHO-606 cells with and without FX, and in the CHO-745 cells with no heparan chains, the uptake of particles was very low under both conditions. N = 2 biological replicates. ( B ). The CHO cell lines were infected with 1000 vpc of each species D adenovirus. After 3 hpi no effect of FX was seen either in wild type HSPG CHO cells or in the HSPG depleted cells. Mean ± SEM of n = 3 biological replicates is shown.

    Techniques Used: Infection, Isolation

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    New England Biolabs monarch genomic dna purification kit
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    New England Biolabs monarch genomic dna purification kits
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