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    New England Biolabs hindiii new england biolabs
    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, <t>HindIII</t> sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype
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    New England Biolabs new england biolabs hindiii hf
    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and <t>HindIII.</t> Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.
    New England Biolabs Hindiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Journal: BMC Genomics

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    doi: 10.1186/s12864-016-3331-9

    Figure Lengend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Article Snippet: For floxCol12a1 screening, PCR products were digested with HindIII (NEB).

    Techniques: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques: Mutagenesis

    70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation, Immunostaining, Fluorescence In Situ Hybridization

    Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Isolation

    Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation

    Dynamics of enhancer activity and interactivity between human pluripotent states. (A) Plot showing the number of ROSE-called SEs in naïve and primed PSCs. As illustrated in the diagram, two values are given for shared SEs because a SE in one cell type may overlap with two individually-called SEs in the other cell type. (B) Diagram showing the number of genes that are contacted by SEs in the two pluripotent cell types. Shared genes (orange) are genes that are contacted by SE elements in both naïve and primed PSCs. Naïve-specific genes (blue) and primed-specific genes (red) are contacted by SEs in either naïve or primed PSCs, respectively. (C) Plots showing the log2 FPKM expression of genes that interact with SEs in each cell type (naïve, n=648; primed, n=286; shared, n=174). P-values are derived from a Mann Whitney U test. (D) Plot showing the distribution of ROSE-called enhancers in naïve and primed PSCs. (E) Diagram showing the number of genes that are contacted by enhancers in the two pluripotent cell types. Genes that are also in contact with a SE have been removed from this list of enhancer-interacting genes. (F) Plots showing the log2 FPKM expression of genes that interact with enhancer elements in each cell type (naïve, n=2077; primed, n=1210; shared, n=3158). P-values are derived from a Mann Whitney U test. (G) Genome browser view of the DPPA5 promoter interactomes in naïve (upper) and primed (lower) PSCs. Significant interactions are shown as blue arcs that connect the baited HindIII fragment containing the DPPA5 promoter (shaded in red) with promoter-interacting regions (shaded in green). ChIP-seq (H3K4me1, H3K4me3 and H3K27ac) and RNA-seq tracks are shown. Chromatin states include active chromatin, light green; H3K4me1-only chromatin, dark green; bivalent chromatin, purple; background, grey. ROSE tracks show the location of enhancers (green) and super-enhancers (red), and OSN tracks show the position of shared (orange) and naïve-specific (blue) regions of OSN occupancy. (H) Network graph showing the locations and cell-type-origin of enhancer and SE elements. Colours depict naïve-specific (blue), primed-specific (red) and shared (orange) enhancer and SE elements. Node size represents SE (large nodes) and enhancers (small nodes). Lines represent interactions and are coloured according to the colour of the node of origin. (I-J) Plots show the (I) percent DNA methylation or (J) histone modification levels in naïve and primed PSCs at shared (n=18,735) and cell type-specific active enhancers (naïve, n=26,955; primed, n=34,805). Regions that are in the background chromatin state in both cell types are shown to indicate genome-wide levels (n=467,772). See also Figure S6 .

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Dynamics of enhancer activity and interactivity between human pluripotent states. (A) Plot showing the number of ROSE-called SEs in naïve and primed PSCs. As illustrated in the diagram, two values are given for shared SEs because a SE in one cell type may overlap with two individually-called SEs in the other cell type. (B) Diagram showing the number of genes that are contacted by SEs in the two pluripotent cell types. Shared genes (orange) are genes that are contacted by SE elements in both naïve and primed PSCs. Naïve-specific genes (blue) and primed-specific genes (red) are contacted by SEs in either naïve or primed PSCs, respectively. (C) Plots showing the log2 FPKM expression of genes that interact with SEs in each cell type (naïve, n=648; primed, n=286; shared, n=174). P-values are derived from a Mann Whitney U test. (D) Plot showing the distribution of ROSE-called enhancers in naïve and primed PSCs. (E) Diagram showing the number of genes that are contacted by enhancers in the two pluripotent cell types. Genes that are also in contact with a SE have been removed from this list of enhancer-interacting genes. (F) Plots showing the log2 FPKM expression of genes that interact with enhancer elements in each cell type (naïve, n=2077; primed, n=1210; shared, n=3158). P-values are derived from a Mann Whitney U test. (G) Genome browser view of the DPPA5 promoter interactomes in naïve (upper) and primed (lower) PSCs. Significant interactions are shown as blue arcs that connect the baited HindIII fragment containing the DPPA5 promoter (shaded in red) with promoter-interacting regions (shaded in green). ChIP-seq (H3K4me1, H3K4me3 and H3K27ac) and RNA-seq tracks are shown. Chromatin states include active chromatin, light green; H3K4me1-only chromatin, dark green; bivalent chromatin, purple; background, grey. ROSE tracks show the location of enhancers (green) and super-enhancers (red), and OSN tracks show the position of shared (orange) and naïve-specific (blue) regions of OSN occupancy. (H) Network graph showing the locations and cell-type-origin of enhancer and SE elements. Colours depict naïve-specific (blue), primed-specific (red) and shared (orange) enhancer and SE elements. Node size represents SE (large nodes) and enhancers (small nodes). Lines represent interactions and are coloured according to the colour of the node of origin. (I-J) Plots show the (I) percent DNA methylation or (J) histone modification levels in naïve and primed PSCs at shared (n=18,735) and cell type-specific active enhancers (naïve, n=26,955; primed, n=34,805). Regions that are in the background chromatin state in both cell types are shown to indicate genome-wide levels (n=467,772). See also Figure S6 .

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques: Activity Assay, Expressing, Derivative Assay, MANN-WHITNEY, Chromatin Immunoprecipitation, RNA Sequencing Assay, DNA Methylation Assay, Modification, Genome Wide

    Multi-scale exploration of promoter-interaction data using force-directed network graphs. (A) Representation of PCHi-C data as arc diagrams (upper) and as corresponding network graphs (lower). Interacting HindIII genomic fragments are depicted as nodes that are connected by edges (significant interactions). A combined network graph is created by merging naïve and primed human PSC datasets whilst retaining cell type-specific information. Blue, naïve-specific nodes and edges; red, primed-specific nodes and edges; grey, shared nodes and edges. (B) Canvas produces a force-directed layout of the combined, whole-network graph. Nodes that interact more frequently are pulled closer together, and less interacting nodes are pushed further apart. (C) Differential gene expression (p-adj

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Multi-scale exploration of promoter-interaction data using force-directed network graphs. (A) Representation of PCHi-C data as arc diagrams (upper) and as corresponding network graphs (lower). Interacting HindIII genomic fragments are depicted as nodes that are connected by edges (significant interactions). A combined network graph is created by merging naïve and primed human PSC datasets whilst retaining cell type-specific information. Blue, naïve-specific nodes and edges; red, primed-specific nodes and edges; grey, shared nodes and edges. (B) Canvas produces a force-directed layout of the combined, whole-network graph. Nodes that interact more frequently are pulled closer together, and less interacting nodes are pushed further apart. (C) Differential gene expression (p-adj

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques: Expressing

    Long-range promoter interactions in primed PSCs drive genome conformation changes between pluripotent states. (A) Plot shows the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. Each small circle represents a different sub-network. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The protocadherin ( PCDH ), histone H1 ( HISTH1 ) and ‘most changing’ (containing diverse genes) sub-networks are highlighted in red. (B) Multidimensional scaling representation (MDS) of the ‘most changing’ sub-network plotted using the linear genomic distance between nodes as edge weights. The measured stress score of 0.139 indicates there is a reasonable fit between the linear genomic distances and the spacing of the nodes as determined by MDS ( Kruskal and Wish, 1978 ). (C) Plot shows the distribution of linear genomic distances between interacting nodes in naïve and primed PSCs, binned into long-, mid-, and short-range distances. (D) Chart shows the total number of chromatin loops identified on chromosome 5 in naïve and primed PSCs. (E) Hi-C interaction matrix of chromosome 5 at a resolution of 250 kb with Knight-Ruiz (KR) normalisation; upper right, naïve PSCs; lower left, primed PSCs. Areas of contact enrichment were defined separately for naïve and primed PSCs using HiCCUPS and each cell type-specific set of chromatin interactions are highlighted as a black square on their respective heatmap. The two corner numbers indicates the maximum intensity values for the matrix. The tracks below the Hi-C heatmap show the PCHi-C interactions and ChiCAGO scores over the same region. (F) Heatmap shows the aggregate peak analysis (250 kb resolution) of primed-specific chromatin interactions on chromosome 5 for naïve and primed PSCs. Chromatin interactions > 5 Mb from the diagonal were used for the analysis (n=27 loops out of a total of 76). The ‘peak to lower left’ (P2LL) score denotes the enrichment of the central pixel over the pixels in the lower left quadrant. See also Figures S3 and S4 .

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Long-range promoter interactions in primed PSCs drive genome conformation changes between pluripotent states. (A) Plot shows the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. Each small circle represents a different sub-network. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The protocadherin ( PCDH ), histone H1 ( HISTH1 ) and ‘most changing’ (containing diverse genes) sub-networks are highlighted in red. (B) Multidimensional scaling representation (MDS) of the ‘most changing’ sub-network plotted using the linear genomic distance between nodes as edge weights. The measured stress score of 0.139 indicates there is a reasonable fit between the linear genomic distances and the spacing of the nodes as determined by MDS ( Kruskal and Wish, 1978 ). (C) Plot shows the distribution of linear genomic distances between interacting nodes in naïve and primed PSCs, binned into long-, mid-, and short-range distances. (D) Chart shows the total number of chromatin loops identified on chromosome 5 in naïve and primed PSCs. (E) Hi-C interaction matrix of chromosome 5 at a resolution of 250 kb with Knight-Ruiz (KR) normalisation; upper right, naïve PSCs; lower left, primed PSCs. Areas of contact enrichment were defined separately for naïve and primed PSCs using HiCCUPS and each cell type-specific set of chromatin interactions are highlighted as a black square on their respective heatmap. The two corner numbers indicates the maximum intensity values for the matrix. The tracks below the Hi-C heatmap show the PCHi-C interactions and ChiCAGO scores over the same region. (F) Heatmap shows the aggregate peak analysis (250 kb resolution) of primed-specific chromatin interactions on chromosome 5 for naïve and primed PSCs. Chromatin interactions > 5 Mb from the diagonal were used for the analysis (n=27 loops out of a total of 76). The ‘peak to lower left’ (P2LL) score denotes the enrichment of the central pixel over the pixels in the lower left quadrant. See also Figures S3 and S4 .

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques: Hi-C

    Assigning ChromHMM states to chromatin interacting regions (A) Heatmap shows the number of interacting HindIII fragments that were assigned to each of the ChromHMM-defined chromatin states in both cell types. Note the higher number of bivalently-marked (H3K27me3 and H3K4me1/3) interacting regions in primed compared to naïve PSCs. (B) Chart shows the total number of interacting HindIII fragments for each of the ChromHMM states. (C) Sankey plot reveals the chromatin state composition for the 617 HindIII interacting regions in naïve PSCs that were defined by ChromHMM as being in a ‘mixed’ chromatin state. The left column shows the 617 mixed chromatin state regions and the right column shows the breakdown of individual chromatin states for each of the regions, represented by the different colour bars. Approximately half of the mixed state HindIII fragments contain signatures of active (H3K4me3, green), bivalent (H3K27me3 and H3K4me1/3, orange) and Polycomb (H3K27me3-only, red) chromatin. (D) Heatmap shows the difference in HindIII fragment interaction frequency between cell types as a function of the chromatin state of the interacting regions (rows) and the linear interaction distance (columns, binned distances). Interacting regions that are engaged in long-range promoter interactions, defined as > 1Mb, are highlighted by the dashed box. Nearly all (98%) of the long-range interactions were associated with bivalently-marked promoters and these regions have a higher interaction frequency in primed compared to naïve PSCs.

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Assigning ChromHMM states to chromatin interacting regions (A) Heatmap shows the number of interacting HindIII fragments that were assigned to each of the ChromHMM-defined chromatin states in both cell types. Note the higher number of bivalently-marked (H3K27me3 and H3K4me1/3) interacting regions in primed compared to naïve PSCs. (B) Chart shows the total number of interacting HindIII fragments for each of the ChromHMM states. (C) Sankey plot reveals the chromatin state composition for the 617 HindIII interacting regions in naïve PSCs that were defined by ChromHMM as being in a ‘mixed’ chromatin state. The left column shows the 617 mixed chromatin state regions and the right column shows the breakdown of individual chromatin states for each of the regions, represented by the different colour bars. Approximately half of the mixed state HindIII fragments contain signatures of active (H3K4me3, green), bivalent (H3K27me3 and H3K4me1/3, orange) and Polycomb (H3K27me3-only, red) chromatin. (D) Heatmap shows the difference in HindIII fragment interaction frequency between cell types as a function of the chromatin state of the interacting regions (rows) and the linear interaction distance (columns, binned distances). Interacting regions that are engaged in long-range promoter interactions, defined as > 1Mb, are highlighted by the dashed box. Nearly all (98%) of the long-range interactions were associated with bivalently-marked promoters and these regions have a higher interaction frequency in primed compared to naïve PSCs.

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques:

    Changes in promoter-interaction frequency and transcriptional levels in four gene clusters (A) Box plots show the number of significant interactions at HindIII fragments located within the HISTH1 , PCDH , KRT and Olfactory gene clusters. (B) Volcano plots show the transcriptional changes between naïve and primed PSCs for genes within the HISTH1 , PCDH , KRT and Olfactory gene clusters. Each dot represents a different gene. Genes coloured in red are differentially expressed between naïve and primed PSCs (log10 fold change > 1.5 or > −1.5 and with an adjusted p-value

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Changes in promoter-interaction frequency and transcriptional levels in four gene clusters (A) Box plots show the number of significant interactions at HindIII fragments located within the HISTH1 , PCDH , KRT and Olfactory gene clusters. (B) Volcano plots show the transcriptional changes between naïve and primed PSCs for genes within the HISTH1 , PCDH , KRT and Olfactory gene clusters. Each dot represents a different gene. Genes coloured in red are differentially expressed between naïve and primed PSCs (log10 fold change > 1.5 or > −1.5 and with an adjusted p-value

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques:

    Long-range promoter interactions create large sub-networks in primed PSCs (A) Scatter plots show the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The HOXA , HOXD , NKX and HISTH1 sub-networks are highlighted. Sub-networks are coloured according to their number of long-range promoter interactions. Note the increased number of long-range promoter interactions within most sub-networks in primed (right) compared to naïve (left) PSCs. (B) Genome browser tracks show the PCHi-C interactions and CHiCAGO scores in naïve and primed PSCs for the HOXA , HOXD , NKX and HISTH1 sub-networks. (C) Dot plots show that the high number of long-range promoter interactions in primed PSCs is independent of the applied CHiCAGO threshold. Each dot represents a PCHi-C interaction, positioned according to the linear genomic distance of the interaction (x-axis) and the assigned CHiCAGO score (y-axis). Black dots show the interactions obtained when applying a CHiCAGO score of > 5 (the threshold used for constructing the network graph) and red dots show the interactions when using a relaxed CHiCAGO score of between 3 and 5. Primed PSCs have more long-range promoter interactions (shaded area; defined as > 1Mb) compared to naïve PSCs when either CHiCAGO threshold score is applied.

    Journal: bioRxiv

    Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

    doi: 10.1101/2019.12.13.875286

    Figure Lengend Snippet: Long-range promoter interactions create large sub-networks in primed PSCs (A) Scatter plots show the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The HOXA , HOXD , NKX and HISTH1 sub-networks are highlighted. Sub-networks are coloured according to their number of long-range promoter interactions. Note the increased number of long-range promoter interactions within most sub-networks in primed (right) compared to naïve (left) PSCs. (B) Genome browser tracks show the PCHi-C interactions and CHiCAGO scores in naïve and primed PSCs for the HOXA , HOXD , NKX and HISTH1 sub-networks. (C) Dot plots show that the high number of long-range promoter interactions in primed PSCs is independent of the applied CHiCAGO threshold. Each dot represents a PCHi-C interaction, positioned according to the linear genomic distance of the interaction (x-axis) and the assigned CHiCAGO score (y-axis). Black dots show the interactions obtained when applying a CHiCAGO score of > 5 (the threshold used for constructing the network graph) and red dots show the interactions when using a relaxed CHiCAGO score of between 3 and 5. Primed PSCs have more long-range promoter interactions (shaded area; defined as > 1Mb) compared to naïve PSCs when either CHiCAGO threshold score is applied.

    Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

    Techniques:

    Effects of ssl2-508 on gene looping. A , schematic depiction of SEN1 , BLM10 , and HEM3 genes, including the positions of the HindIII sites and the P1 and T1 primer pairs. Approximate length of each ORF is indicated as kb. For description of the 3C assay,

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanism of Start Site Selection by RNA Polymerase II

    doi: 10.1074/jbc.M111.281576

    Figure Lengend Snippet: Effects of ssl2-508 on gene looping. A , schematic depiction of SEN1 , BLM10 , and HEM3 genes, including the positions of the HindIII sites and the P1 and T1 primer pairs. Approximate length of each ORF is indicated as kb. For description of the 3C assay,

    Article Snippet: Chromatin was extracted and digested overnight at 37 °C with gentle shaking in the presence of the restriction enzyme HindIII (New England Biolabs).

    Techniques:

    Gene-rich and gene-poor regions are organized into distinct TADs at the EDC locus in keratinocytes. (a) Schematic structure of the 5.3 Mb genomic region containing the EDC locus on mouse chromosome 3 analysed using 5C technology in this manuscript (mm9/chr.3:89,900,000–95,200,000). (b) Alternating 5C probe design for the unique HindIII sites in the interrogated genomic regions. The position of the restriction sites interrogated by the forward primers are shown in blue, interrogated by the reverse primers are shown in red and the site for which the primers could not be designed are shown in green. (c) Heatmaps representing raw 5C data for both KC replicates. Reverse probes are plotted as columns and the forward probes as rows. Pearson’s correlation coefficient is also shown. (d) Heatmap representing the 5C data after the normalization and binning (bin size 150 kb, step size 15kb) in KCs. The position of TAD border midpoints (average for the midpoints calculated based on the insulation index analysis in two replicates independently) are identified by green lines under the heatmaps. Note the high frequency of the spatial contacts between the gene-poor TADs 2 and 5 (indicated by dashed rectangle on the heat map). The position of the regions covered by the BAC fish probes used in these studies, schematic map of the studied locus and insulation indexes profiles for two 5C library replicates are also shown. (e) Multi-colour 3D FISH analysis with BAC probes A (located at the 5’ border of TAD3, B (located at the 3’ border of TAD4) and C (located within TAD4) (left) , or with BAC probe D (located within gene-poor TAD2) and E (located within gene-poor TAD5) (right) in basal epidermal keratinocytes. Representative single optical sections are shown. Scale bars are 2μm. (f) Box plots showing median, 25% quartile, 75% quartile with whiskers indicating maximum and minimum for spatial distances between the centres of the regions covered by probes A and B, probes B and C, as well as probes D and E before (in μm) and after normalization to the average nuclear radius (in % of average nuclear radius) in basal epidermal keratinocytes in situ . The distances between the centres of the regions covered by the probes A and B (located within TAD3) are significantly shorter than the distances between loci covered by the probes B and C (located within TAD4). The indicated p-values for pair-wise comparison are calculated using Mann-Whitney U-test, n = 60 alleles for each interrogated locus. The distances between the centres of the regions covered by the probes D and E (located in the gene poor TADs 2 and 5 respectively) are similar to the much closer regions covered by the probes B and C (located in the adjacent gene-rich TAD3 and TAD4).

    Journal: PLoS Genetics

    Article Title: 5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells

    doi: 10.1371/journal.pgen.1006966

    Figure Lengend Snippet: Gene-rich and gene-poor regions are organized into distinct TADs at the EDC locus in keratinocytes. (a) Schematic structure of the 5.3 Mb genomic region containing the EDC locus on mouse chromosome 3 analysed using 5C technology in this manuscript (mm9/chr.3:89,900,000–95,200,000). (b) Alternating 5C probe design for the unique HindIII sites in the interrogated genomic regions. The position of the restriction sites interrogated by the forward primers are shown in blue, interrogated by the reverse primers are shown in red and the site for which the primers could not be designed are shown in green. (c) Heatmaps representing raw 5C data for both KC replicates. Reverse probes are plotted as columns and the forward probes as rows. Pearson’s correlation coefficient is also shown. (d) Heatmap representing the 5C data after the normalization and binning (bin size 150 kb, step size 15kb) in KCs. The position of TAD border midpoints (average for the midpoints calculated based on the insulation index analysis in two replicates independently) are identified by green lines under the heatmaps. Note the high frequency of the spatial contacts between the gene-poor TADs 2 and 5 (indicated by dashed rectangle on the heat map). The position of the regions covered by the BAC fish probes used in these studies, schematic map of the studied locus and insulation indexes profiles for two 5C library replicates are also shown. (e) Multi-colour 3D FISH analysis with BAC probes A (located at the 5’ border of TAD3, B (located at the 3’ border of TAD4) and C (located within TAD4) (left) , or with BAC probe D (located within gene-poor TAD2) and E (located within gene-poor TAD5) (right) in basal epidermal keratinocytes. Representative single optical sections are shown. Scale bars are 2μm. (f) Box plots showing median, 25% quartile, 75% quartile with whiskers indicating maximum and minimum for spatial distances between the centres of the regions covered by probes A and B, probes B and C, as well as probes D and E before (in μm) and after normalization to the average nuclear radius (in % of average nuclear radius) in basal epidermal keratinocytes in situ . The distances between the centres of the regions covered by the probes A and B (located within TAD3) are significantly shorter than the distances between loci covered by the probes B and C (located within TAD4). The indicated p-values for pair-wise comparison are calculated using Mann-Whitney U-test, n = 60 alleles for each interrogated locus. The distances between the centres of the regions covered by the probes D and E (located in the gene poor TADs 2 and 5 respectively) are similar to the much closer regions covered by the probes B and C (located in the adjacent gene-rich TAD3 and TAD4).

    Article Snippet: To each aliquot of solubilized chromatin 800 U of HindIII enzyme (New England Biolabs) was added and the digestion was performed overnight at 37°C with shaking.

    Techniques: BAC Assay, Fluorescence In Situ Hybridization, In Situ, MANN-WHITNEY

    Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Journal: International Journal of Molecular Sciences

    Article Title: Production and Evaluation of Virus-Like Particles Displaying Immunogenic Epitopes of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

    doi: 10.3390/ijms16048382

    Figure Lengend Snippet: Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Article Snippet: The 585-bp insert release (Lane 2, A) and the pET28a vector digested with NheI and HindIII enzymes (Lane 5, A) were cloned using T4 DNA ligase enzyme (NEB, Ipswich, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Sequencing

    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Journal: bioRxiv

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    doi: 10.1101/2020.05.20.105924

    Figure Lengend Snippet: ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Article Snippet: Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes.

    Techniques: Polymerase Chain Reaction, Produced, Southern Blot, Sequencing, Plasmid Preparation

    Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Journal: Human genetics

    Article Title: 3C-PCR: A novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants

    doi: 10.1007/s00439-017-1853-0

    Figure Lengend Snippet: Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Article Snippet: Chromatin was digested with Hin dIII-HF (NEB), ligated with T4 DNA ligase (NEB), and reverse crosslinked by incubation with Proteinase K (NEB) and RNase A (EMD Millipore).

    Techniques: Nested PCR, Ligation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Amplification