r0543  (New England Biolabs)


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    Name:
    DpnII
    Description:
    DpnII 5 000 units
    Catalog Number:
    R0543L
    Price:
    290
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    New England Biolabs r0543
    DpnII
    DpnII 5 000 units
    https://www.bioz.com/result/r0543/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r0543 - by Bioz Stars, 2021-05
    99/100 stars

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    Related Articles

    Polymerase Chain Reaction:

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: Single GFP+ iPSCs were maintained in mTeSR1 and allowed to grow into colonies until manually picking for DNA extraction with Quick Extract - DNA Extraction Solution (Epicentre). .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

    Amplification:

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: Single GFP+ iPSCs were maintained in mTeSR1 and allowed to grow into colonies until manually picking for DNA extraction with Quick Extract - DNA Extraction Solution (Epicentre). .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

    Clone Assay:

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: Single GFP+ iPSCs were maintained in mTeSR1 and allowed to grow into colonies until manually picking for DNA extraction with Quick Extract - DNA Extraction Solution (Epicentre). .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Sequencing:

    Article Title: Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing
    Article Snippet: Preparation of N6-methyladenine-containing DNA for sequencing was performed using a previously reported procedure ( ) using primers in with the following modifications: 5 μg of genomic DNA from N. crassa strains expressing a Dam fusion was digested with 1 μL of DpnI (NEB, 20 units/μL); ligation to primer 5050 was carried out overnight at 16 °C; amplification reactions of ligated DNA with primer 5051 were performed in triplicate with 5 μL dNTPs, and an additional PCR cycle was added to the 2nd and 3rd phase of the PCR protocol (4 and 18 cycles respectively); 3 μg of pooled, amplified DNA was sheared using a Bioruptor (Diagenode) twice on high for 10 minutes (30 seconds on/off) at 4 °C; biotinylated DNA was purified using 250 μL slurry of streptavidin-agarose beads (Sigma). .. DNA was cleaved from the beads with DpnII and libraries were prepared for sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs). .. False perithecia assay and image analysisTo assay of the development of false perithecia, strains were grown on modified Vogel’s media( ) as described above without a fertilizing strain.

    Hybridization:

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

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    • dpnii  (New England Biolabs)
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    New England Biolabs dpnii
    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from <t>DpnII-resistant</t> prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by <t>DpnI</t> (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Dpnii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dpnii/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Journal: Nature Communications

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    doi: 10.1038/ncomms12743

    Figure Lengend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Article Snippet: Non-DpnI-restricted DNA was cut with DpnII (New England Biolabs) for 1 h at 37 °C.

    Techniques: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    EPR-1 associates with H3K27 methylation in vivo and in vitro . a , Scatter plot showing the correlation of levels of H3K27me2/3 and GFP-EPR-1 WT for all genes (black dots), as determined by ChIP-seq. Line of best fit displayed in red (R 2 =0.8675). b , Western blot shows GFP-EPR-1 expression in the indicated strains. Phosphoglycerate kinase (PGK) is used as a loading control. Each genotype (except wild type, negative control) was run in biological duplicate and repeated. c , Quantification of the GFP band intensity averaged over 4 biological replicates. The intensities are relative to the corresponding PGK band and normalized to the wild-type average from the same blot. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units). d , Box and whisker plot of normalized GFP-EPR-1 ChIP-seq coverage from epr-1 WT , epr-1 PHD and epr-1 BAH strains is shown for the indicated regions of the genome. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. e , Box and whisker plot of normalized EPR-1 DamID-seq coverage from epr-1 WT , epr-1 PHD and epr-1 WT ; Δeed strains is shown for the indicated regions of the genome. Reads have been corrected for the frequency of GATC sites. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. f , DamID Southern blot of genomic DNA from the indicated strains digested with DpnI (DI), DpnII (DII) or left undigested (-). DpnII, which digests GATC sites without methylated adenines, reveals pattern of complete digestion in wild type. DpnI, which fails to digest GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions ( NCU05173 and Tel VIIL, H3K27-methylated; his-3 , euchromatin). Ethidium bromide (EtBr) shows total DNA. Biological replicates are shown. g , An overlay of 1 H- 15 N HSQC spectra of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. h , An overlay of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion with the 15 N-labelled HIS-SUMO alone. Boxed areas are shown enlarged in Fig. 5d . Spectra are color coded as indicated.

    Journal: bioRxiv

    Article Title: Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing

    doi: 10.1101/868117

    Figure Lengend Snippet: EPR-1 associates with H3K27 methylation in vivo and in vitro . a , Scatter plot showing the correlation of levels of H3K27me2/3 and GFP-EPR-1 WT for all genes (black dots), as determined by ChIP-seq. Line of best fit displayed in red (R 2 =0.8675). b , Western blot shows GFP-EPR-1 expression in the indicated strains. Phosphoglycerate kinase (PGK) is used as a loading control. Each genotype (except wild type, negative control) was run in biological duplicate and repeated. c , Quantification of the GFP band intensity averaged over 4 biological replicates. The intensities are relative to the corresponding PGK band and normalized to the wild-type average from the same blot. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units). d , Box and whisker plot of normalized GFP-EPR-1 ChIP-seq coverage from epr-1 WT , epr-1 PHD and epr-1 BAH strains is shown for the indicated regions of the genome. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. e , Box and whisker plot of normalized EPR-1 DamID-seq coverage from epr-1 WT , epr-1 PHD and epr-1 WT ; Δeed strains is shown for the indicated regions of the genome. Reads have been corrected for the frequency of GATC sites. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. f , DamID Southern blot of genomic DNA from the indicated strains digested with DpnI (DI), DpnII (DII) or left undigested (-). DpnII, which digests GATC sites without methylated adenines, reveals pattern of complete digestion in wild type. DpnI, which fails to digest GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions ( NCU05173 and Tel VIIL, H3K27-methylated; his-3 , euchromatin). Ethidium bromide (EtBr) shows total DNA. Biological replicates are shown. g , An overlay of 1 H- 15 N HSQC spectra of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. h , An overlay of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion with the 15 N-labelled HIS-SUMO alone. Boxed areas are shown enlarged in Fig. 5d . Spectra are color coded as indicated.

    Article Snippet: DNA was cleaved from the beads with DpnII and libraries were prepared for sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs).

    Techniques: Methylation, In Vivo, In Vitro, Chromatin Immunoprecipitation, Western Blot, Expressing, Negative Control, Standard Deviation, Whisker Assay, Southern Blot

    MIAA identifies global influence of GC-content and differentially accessible motifs. A) GC-content observed to be correlated with accessibility in both stem and endoderm cells from positive (universally opening) and negative (universally closing) control sequences. B) GC-content correlated with accessibility in random DNA phrases. The regression model was trained on MIAA Dpn proportions with GC-content, replicate, and cell type-specific effects of 20 motifs and 26 motif pairs as features, and predicts well on (C) training data (n = 21,420) and (D) held-out test data (n = 4,404). The correlation reported is the Pearson correlation coefficient (r). E) Regression weights of individual motifs and motif pairs in stem and definitive endoderm cells. Hierarchical clustering of regression weights followed by motif enrichment recovers clusters representing cell type-specific transcription factor DNA binding motifs. F) Example of individual motifs (left, middle) which alone do not differentially open chromatin, but differentially open chromatin stem cells in combination (right). Each dot represents the average DpnII read proportion of an individual phrase, compared to shuffled controls (CTRL). Significance computed by paired t-test.

    Journal: bioRxiv

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    doi: 10.1101/2020.03.02.973396

    Figure Lengend Snippet: MIAA identifies global influence of GC-content and differentially accessible motifs. A) GC-content observed to be correlated with accessibility in both stem and endoderm cells from positive (universally opening) and negative (universally closing) control sequences. B) GC-content correlated with accessibility in random DNA phrases. The regression model was trained on MIAA Dpn proportions with GC-content, replicate, and cell type-specific effects of 20 motifs and 26 motif pairs as features, and predicts well on (C) training data (n = 21,420) and (D) held-out test data (n = 4,404). The correlation reported is the Pearson correlation coefficient (r). E) Regression weights of individual motifs and motif pairs in stem and definitive endoderm cells. Hierarchical clustering of regression weights followed by motif enrichment recovers clusters representing cell type-specific transcription factor DNA binding motifs. F) Example of individual motifs (left, middle) which alone do not differentially open chromatin, but differentially open chromatin stem cells in combination (right). Each dot represents the average DpnII read proportion of an individual phrase, compared to shuffled controls (CTRL). Significance computed by paired t-test.

    Article Snippet: Genomic DNA library preparation was performed by performing DpnI and DpnII digests using the following conditions: DpnI digest: 10 ug genomic DNA + 10 uL CutSmart Buffer (New England Biolabs) + 2 uL DpnI (New England Biolabs) + up to 100 uL water.

    Techniques: Binding Assay

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Journal: bioRxiv

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    doi: 10.1101/2020.03.02.973396

    Figure Lengend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Article Snippet: Genomic DNA library preparation was performed by performing DpnI and DpnII digests using the following conditions: DpnI digest: 10 ug genomic DNA + 10 uL CutSmart Buffer (New England Biolabs) + 2 uL DpnI (New England Biolabs) + up to 100 uL water.

    Techniques: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    Nucleolar-DamID identifies NADs A. Chromosomal view of NADs, LADs ( Peric-Hupkes et al., 2010 ), A and B compartments ( Dalcher et al., 2020 ), and early and late replicating DNA ( Marchal et al., 2018 ) in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS and H2B-Dam. iNAD: regions not contacting the nucleolus. iLAD: regions not contacting the NL. Chromosomes 2 and 18 are shown. B. NAD coverage. Bars represent NAD coverage values of each mouse chromosome. Red lines showed NAD coverage values in the centromere-proximal end of each chromosome. Chromosome bearing rRNA genes are depicted with orange numbers and bars. Dotted line shows NAD whole genome coverage. C. Nucleolar H2B-Dam chromosomal interaction maps. NAD-only and NAD/LAD regions were represented with orange and grey colors, respectively. Chromosome bearing rRNA genes are depicted with orange numbers. D. Genomic contacts with rRNA genes (rDNA contacts) obtained from published HiC maps ( Bonev et al., 2017 ). Data represent the proportion of identified unique contacts for each chromosome. E. Representative images showing normalized count score of rDNA contacts obtained from the HiC-rDNA on chromosome 9, which does not harbor rRNA genes, and chromosome 18, which contains rRNA genes. F. Distribution of rDNA contacts for each chromosome. Values represent the proportion of the number of unique contacts for each chromosome quintile. Statistical significance ( P -values) for the experiments was calculated using the unpaired two-tailed t-test (****

    Journal: bioRxiv

    Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

    doi: 10.1101/2020.11.17.386797

    Figure Lengend Snippet: Nucleolar-DamID identifies NADs A. Chromosomal view of NADs, LADs ( Peric-Hupkes et al., 2010 ), A and B compartments ( Dalcher et al., 2020 ), and early and late replicating DNA ( Marchal et al., 2018 ) in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS and H2B-Dam. iNAD: regions not contacting the nucleolus. iLAD: regions not contacting the NL. Chromosomes 2 and 18 are shown. B. NAD coverage. Bars represent NAD coverage values of each mouse chromosome. Red lines showed NAD coverage values in the centromere-proximal end of each chromosome. Chromosome bearing rRNA genes are depicted with orange numbers and bars. Dotted line shows NAD whole genome coverage. C. Nucleolar H2B-Dam chromosomal interaction maps. NAD-only and NAD/LAD regions were represented with orange and grey colors, respectively. Chromosome bearing rRNA genes are depicted with orange numbers. D. Genomic contacts with rRNA genes (rDNA contacts) obtained from published HiC maps ( Bonev et al., 2017 ). Data represent the proportion of identified unique contacts for each chromosome. E. Representative images showing normalized count score of rDNA contacts obtained from the HiC-rDNA on chromosome 9, which does not harbor rRNA genes, and chromosome 18, which contains rRNA genes. F. Distribution of rDNA contacts for each chromosome. Values represent the proportion of the number of unique contacts for each chromosome quintile. Statistical significance ( P -values) for the experiments was calculated using the unpaired two-tailed t-test (****

    Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

    Techniques: Two Tailed Test

    Distinct layers of repressive chromatin states distinguish genomic domains according to their interaction with the nucleolus, nuclear lamina, or both A. Venn diagram showing the proportion of NAD-only and NAD/LAD regions in NADs identified by Nucleolar-DamID. B. Venn diagram showing the proportion of LAD-only and NAD/LAD regions in LADs of ESCs. C. Lamin B1-DamID scores plotted over NAD-only and NAD/LAD boundaries in ESCs. D. Gene density of total genome, NAD subclasses, and LAD-only. E. Amounts (%) of NAD-only, LAD-only and NAD/LAD in A and B compartments. F. Amounts (%) of early and late replicating DNA of NAD-only, LAD-only and NAD/LAD sequences. G. Expression values (RPKM) of genes within A compartment (A Comp.), NAD-only, LAD-only, and NAD/LAD. Statistical significance ( P -values) was calculated using the unpaired two-tailed t-test (***

    Journal: bioRxiv

    Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

    doi: 10.1101/2020.11.17.386797

    Figure Lengend Snippet: Distinct layers of repressive chromatin states distinguish genomic domains according to their interaction with the nucleolus, nuclear lamina, or both A. Venn diagram showing the proportion of NAD-only and NAD/LAD regions in NADs identified by Nucleolar-DamID. B. Venn diagram showing the proportion of LAD-only and NAD/LAD regions in LADs of ESCs. C. Lamin B1-DamID scores plotted over NAD-only and NAD/LAD boundaries in ESCs. D. Gene density of total genome, NAD subclasses, and LAD-only. E. Amounts (%) of NAD-only, LAD-only and NAD/LAD in A and B compartments. F. Amounts (%) of early and late replicating DNA of NAD-only, LAD-only and NAD/LAD sequences. G. Expression values (RPKM) of genes within A compartment (A Comp.), NAD-only, LAD-only, and NAD/LAD. Statistical significance ( P -values) was calculated using the unpaired two-tailed t-test (***

    Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

    Techniques: Expressing, Two Tailed Test

    Nucleolar-DamID in ESCs A. Pearson correlation of Nucleolar-DamID experiments in ESCs and NPCs. B. Violin plot showing distribution of NAD length. C. Chromosomal view of NADs in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS vs. H2B-Dam. iNAD: inter-NAD regions. iLAD: inter-LAD regions. Pink bars correspond to sequence found in nucleolar hubs using SPRITE method ( Quinodoz et al., 2018 ). D. Upper panel. NAD and LAD composition of chromosome 14 and FISH probe corresponding to a strong LAD (blue bar). Lower panel. Representative images from immunofluorescence for nucleolin (green) combined with DNA FISH using probes corresponding to LAD-only region of chromosome 14 (red), and DAPI (blue). The signal of the LAD probe was located close to NL in the majority of analyzed ESCs (27/33).

    Journal: bioRxiv

    Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

    doi: 10.1101/2020.11.17.386797

    Figure Lengend Snippet: Nucleolar-DamID in ESCs A. Pearson correlation of Nucleolar-DamID experiments in ESCs and NPCs. B. Violin plot showing distribution of NAD length. C. Chromosomal view of NADs in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS vs. H2B-Dam. iNAD: inter-NAD regions. iLAD: inter-LAD regions. Pink bars correspond to sequence found in nucleolar hubs using SPRITE method ( Quinodoz et al., 2018 ). D. Upper panel. NAD and LAD composition of chromosome 14 and FISH probe corresponding to a strong LAD (blue bar). Lower panel. Representative images from immunofluorescence for nucleolin (green) combined with DNA FISH using probes corresponding to LAD-only region of chromosome 14 (red), and DAPI (blue). The signal of the LAD probe was located close to NL in the majority of analyzed ESCs (27/33).

    Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

    Techniques: Sequencing, Fluorescence In Situ Hybridization, Immunofluorescence