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  • 99
    Thermo Fisher 3730xl dna analyzer
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna sequence
    Dna Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc rna sequencing
    Genome-wide evaluation of mRNA stability states of expressed genes in RA <t>FLS.</t> (a-c), Gene tracks showing sequencing reads from <t>RNA</t> sequencing mapped to CCL20 (a), JUN (b) and IRF1 (c) genes. The sequencing reads after TNF stimulation for 1 hour without (blue) or with Act D (orange) are shown. (d), Stacked bar graphs illustrating the mRNA stability states of genes expressed in unstimulated (Control) and TNF-stimulated FLS (1, 3, 24 and 72 hours of TNF stimulation). The mRNA stability status was calculated as the ratio of expression levels at the TNF+Act D condition divided to the expression levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The expressed genes were classified into five groups with distinct stability states and the size of each group is represented as % of total number of expressed genes for each condition.
    Rna Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Biotechnology Information dna sequences
    (a) Haplotype network of the combined three cp <t>DNA</t> ( <t>psb</t> A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F); (b) Haplotype network of the combined four DNA ( ITS 2, psb A‐ trn H, mat K, and rbc L) regions using median joining method. Blue dots stand for Tripterygium wilfordii , red dots stand for Tripterygium hypoglaucum , green dots stands for Tripterygium regelii , and black dots stand for Celastrus orbiculatus
    Dna Sequences, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 94/100, based on 2081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioedit Company dna sequences
    Titration of the <t>DNA</t> donor and integrase concentrations for the strand transfer assay. (A) Purification of <t>HIV-O</t> recombinant integrase proteins from clades A (IN-O/A) and B (IN-O/B) and a divergent strain (IN-O/Div) and HIV-M recombinant integrase protein (IN-M/B). SDS-PAGE analysis showing that the integrase proteins resolved predominantly in a high-density band (35.6 kDa); the two bands at the bottom represent nonspecifically cleaved forms of the protein. (B) Calibration of the DNA donor mimicking LTR-O using various concentrations, ranging from 0 to 2,400 nM; (C) calibration of the different HIV-O integrases, IN-O/A (clade A), IN-O/B (clade B), and IN-O/Div (divergent strain), and the HIV-M integrase, IN-M/B (HIV-M subtype B), using various concentrations, ranging from 0 to 1,600 nM.
    Dna Sequences, supplied by Bioedit Company, used in various techniques. Bioz Stars score: 93/100, based on 1763 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa smart seq v4 ultra low input rna kit
    Titration of the <t>DNA</t> donor and integrase concentrations for the strand transfer assay. (A) Purification of <t>HIV-O</t> recombinant integrase proteins from clades A (IN-O/A) and B (IN-O/B) and a divergent strain (IN-O/Div) and HIV-M recombinant integrase protein (IN-M/B). SDS-PAGE analysis showing that the integrase proteins resolved predominantly in a high-density band (35.6 kDa); the two bands at the bottom represent nonspecifically cleaved forms of the protein. (B) Calibration of the DNA donor mimicking LTR-O using various concentrations, ranging from 0 to 2,400 nM; (C) calibration of the different HIV-O integrases, IN-O/A (clade A), IN-O/B (clade B), and IN-O/Div (divergent strain), and the HIV-M integrase, IN-M/B (HIV-M subtype B), using various concentrations, ranging from 0 to 1,600 nM.
    Smart Seq V4 Ultra Low Input Rna Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher abi prism 377 dna sequencer
    Titration of the <t>DNA</t> donor and integrase concentrations for the strand transfer assay. (A) Purification of <t>HIV-O</t> recombinant integrase proteins from clades A (IN-O/A) and B (IN-O/B) and a divergent strain (IN-O/Div) and HIV-M recombinant integrase protein (IN-M/B). SDS-PAGE analysis showing that the integrase proteins resolved predominantly in a high-density band (35.6 kDa); the two bands at the bottom represent nonspecifically cleaved forms of the protein. (B) Calibration of the DNA donor mimicking LTR-O using various concentrations, ranging from 0 to 2,400 nM; (C) calibration of the different HIV-O integrases, IN-O/A (clade A), IN-O/B (clade B), and IN-O/Div (divergent strain), and the HIV-M integrase, IN-M/B (HIV-M subtype B), using various concentrations, ranging from 0 to 1,600 nM.
    Abi Prism 377 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript dna sequences
    Aptamer Binding Selectively Reduces Mutant <t>Huntingtin’s</t> PRC2-Stimulating Activity (A) The affinities of Q78-huntingtin alone and huntingtin-aptamer complexes to PRC2 were compared by immunoprecipitation with an anti-EZH2 antibody and an anti-huntingtin antibody (HP-1), followed by immunoblotting for huntingtin and EZH2. The experiment was repeated three times. (B) Autoradiogram of bands of 3 H-methyl histone H3 produced by PRC2 in the absence and presence of mutant-huntingtin-preferring <t>DNA</t> aptamers. Bottom: the bar graph of the band densitometry results, showing that aptamer binding significantly reduced the ability of mutant huntingtin to enhance basal PRC2 activity. Mean band intensities were measured from three independent experiments. Asterisks indicate statistically significant differences compared with unbound Q78-huntingtin. Error bars represent SEM. *p
    Dna Sequences, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 3730 dna sequencer
    Aptamer Binding Selectively Reduces Mutant <t>Huntingtin’s</t> PRC2-Stimulating Activity (A) The affinities of Q78-huntingtin alone and huntingtin-aptamer complexes to PRC2 were compared by immunoprecipitation with an anti-EZH2 antibody and an anti-huntingtin antibody (HP-1), followed by immunoblotting for huntingtin and EZH2. The experiment was repeated three times. (B) Autoradiogram of bands of 3 H-methyl histone H3 produced by PRC2 in the absence and presence of mutant-huntingtin-preferring <t>DNA</t> aptamers. Bottom: the bar graph of the band densitometry results, showing that aptamer binding significantly reduced the ability of mutant huntingtin to enhance basal PRC2 activity. Mean band intensities were measured from three independent experiments. Asterisks indicate statistically significant differences compared with unbound Q78-huntingtin. Error bars represent SEM. *p
    Abi 3730 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genewiz dna sequence analysis
    The <t>bbb22</t> gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. <t>DNA</t> was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.
    Dna Sequence Analysis, supplied by Genewiz, used in various techniques. Bioz Stars score: 93/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher automatic dna sequencer
    The <t>bbb22</t> gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. <t>DNA</t> was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.
    Automatic Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins dna sequences
    Telomere <t>DNA</t> G-quadruplex unfolding by arginine to alanine mutants of <t>UP1+RGG</t> monitored using CD spectroscopy. ( A, B ) Unfolding of K + form of Tel22 G-quadruplex DNA by TriRGG (A) and AllRGG (B) mutants. The G-quadruplex DNA was titrated with increasing molar excess of proteins. The black arrows in the spectra indicate the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( C ) The normalized ellipticity at 295 nm of Tel22 at the final titration step (at 1:6 molar ratio of DNA to protein) for UP1, AllRGG, TriRGG and UP1+RGG showing the relative foldedness of the G-quadruplex structure.
    Dna Sequences, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc illumina dna sequencing
    Relationship between the <t>Illumina</t> <t>DNA-sequencing</t> read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.
    Illumina Dna Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins dna sequencing
    Relationship between the <t>Illumina</t> <t>DNA-sequencing</t> read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.
    Dna Sequencing, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 4234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi prism 3100 dna sequencer
    Relationship between the <t>Illumina</t> <t>DNA-sequencing</t> read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.
    Abi Prism 3100 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 377 dna sequencer
    Relationship between the <t>Illumina</t> <t>DNA-sequencing</t> read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.
    Abi 377 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Genome-wide evaluation of mRNA stability states of expressed genes in RA FLS. (a-c), Gene tracks showing sequencing reads from RNA sequencing mapped to CCL20 (a), JUN (b) and IRF1 (c) genes. The sequencing reads after TNF stimulation for 1 hour without (blue) or with Act D (orange) are shown. (d), Stacked bar graphs illustrating the mRNA stability states of genes expressed in unstimulated (Control) and TNF-stimulated FLS (1, 3, 24 and 72 hours of TNF stimulation). The mRNA stability status was calculated as the ratio of expression levels at the TNF+Act D condition divided to the expression levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The expressed genes were classified into five groups with distinct stability states and the size of each group is represented as % of total number of expressed genes for each condition.

    Journal: PLoS ONE

    Article Title: Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1371/journal.pone.0179762

    Figure Lengend Snippet: Genome-wide evaluation of mRNA stability states of expressed genes in RA FLS. (a-c), Gene tracks showing sequencing reads from RNA sequencing mapped to CCL20 (a), JUN (b) and IRF1 (c) genes. The sequencing reads after TNF stimulation for 1 hour without (blue) or with Act D (orange) are shown. (d), Stacked bar graphs illustrating the mRNA stability states of genes expressed in unstimulated (Control) and TNF-stimulated FLS (1, 3, 24 and 72 hours of TNF stimulation). The mRNA stability status was calculated as the ratio of expression levels at the TNF+Act D condition divided to the expression levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The expressed genes were classified into five groups with distinct stability states and the size of each group is represented as % of total number of expressed genes for each condition.

    Article Snippet: RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts.

    Techniques: Genome Wide, Sequencing, RNA Sequencing Assay, Activated Clotting Time Assay, Expressing

    Scatterplots comparing the expression levels to the mRNA stability states of the expressed genes in RA FLS. Two biological replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1, 3, 24, or 72 hours. Subsequently, actinomycin D (Act D, 10μg/ml) was added for 3 hours to block active transcription and gene expression was measured by RNA sequencing. RPKM values were generated using CuffDiff2. The mRNA stability status was calculated genome-wide as the ratio of RPKM levels at the TNF+Act D condition divided to the RPKM levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The genes expressed at 1 (a), 3 (b), 24 (c), and 72 (d) hours of TNF stimulation were plotted based on their expression levels and the mRNA stability states. Shades of blue represent the region of unstable genes, and shades of red represent the zone of stable genes.

    Journal: PLoS ONE

    Article Title: Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1371/journal.pone.0179762

    Figure Lengend Snippet: Scatterplots comparing the expression levels to the mRNA stability states of the expressed genes in RA FLS. Two biological replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1, 3, 24, or 72 hours. Subsequently, actinomycin D (Act D, 10μg/ml) was added for 3 hours to block active transcription and gene expression was measured by RNA sequencing. RPKM values were generated using CuffDiff2. The mRNA stability status was calculated genome-wide as the ratio of RPKM levels at the TNF+Act D condition divided to the RPKM levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The genes expressed at 1 (a), 3 (b), 24 (c), and 72 (d) hours of TNF stimulation were plotted based on their expression levels and the mRNA stability states. Shades of blue represent the region of unstable genes, and shades of red represent the zone of stable genes.

    Article Snippet: RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts.

    Techniques: Expressing, Derivative Assay, Activated Clotting Time Assay, Blocking Assay, RNA Sequencing Assay, Generated, Genome Wide

    Association of expression kinetics with mRNA stability states of TNF-inducible genes in RA FLS. For (a-b), two biological replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1-72h. Subsequently, Act D (10 μg/ml) was added for 3h and gene expression was measured by RNA sequencing. 386 genes were identified as highly induced (≥5-fold) by TNF at any time point and were clustered into 6 clusters with distinct kinetics of peak expression. (a), Heatmap illustrating the expression kinetics of the 6 clusters (red represents the maximum and blue the minimum expression level across the lane). (b), Stacked bar graphs illustrating the stability states of genes for Cluster 1, Clusters 2 3, Cluster 4, and Clusters 5 6. For (c-f), RA FLS were exposed to a single dose of TNF (10 ng/ml) for 1–72 hours. Primers specific for the eighth intronic region of MMP3 and for the first intronic region of CCL5 were designed to capture primary transcripts (PT) of MMP3 and CCL5 . qPCR was used to measure the levels of PT and total mRNA of MMP3 (c-d) and CCL5 (e-f). Cumulative results from six independent experiments are shown. Values were normalized relative to mRNA for GAPDH and are presented as mean ±SEM.

    Journal: PLoS ONE

    Article Title: Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1371/journal.pone.0179762

    Figure Lengend Snippet: Association of expression kinetics with mRNA stability states of TNF-inducible genes in RA FLS. For (a-b), two biological replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1-72h. Subsequently, Act D (10 μg/ml) was added for 3h and gene expression was measured by RNA sequencing. 386 genes were identified as highly induced (≥5-fold) by TNF at any time point and were clustered into 6 clusters with distinct kinetics of peak expression. (a), Heatmap illustrating the expression kinetics of the 6 clusters (red represents the maximum and blue the minimum expression level across the lane). (b), Stacked bar graphs illustrating the stability states of genes for Cluster 1, Clusters 2 3, Cluster 4, and Clusters 5 6. For (c-f), RA FLS were exposed to a single dose of TNF (10 ng/ml) for 1–72 hours. Primers specific for the eighth intronic region of MMP3 and for the first intronic region of CCL5 were designed to capture primary transcripts (PT) of MMP3 and CCL5 . qPCR was used to measure the levels of PT and total mRNA of MMP3 (c-d) and CCL5 (e-f). Cumulative results from six independent experiments are shown. Values were normalized relative to mRNA for GAPDH and are presented as mean ±SEM.

    Article Snippet: RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts.

    Techniques: Expressing, Derivative Assay, Activated Clotting Time Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Genome-wide identification of transcripts stabilized by TNF in RA FLS. Two biologic replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1 or 72h. Subsequently, Act D was added for 3h and gene expression was measured by RNA sequencing. The degree of TNF-induced mRNA stabilization was calculated as the log 2 difference of TNF+Act D/TNF ratio between 1 and 72h of TNF stimulation and the adjusted p values of TNF-induced stabilization were calculated by RiboDiff. (a), Scatter-plot of the genes displaying TNF-induced mRNA stabilization comparing the degree of mRNA stabilization (y axis) to the adjusted p values of the stabilizing effect of TNF (x-axis). (b), The top 40 genes displaying the highest TNF-induced mRNA stabilization ranked by the degree of stabilization. (c), Enriched biological processes identified by GSEA/MSigDB pathway analysis of the top 10% of the genes (n = 593) displaying the highest degree of TNF-induced mRNA stabilization.

    Journal: PLoS ONE

    Article Title: Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1371/journal.pone.0179762

    Figure Lengend Snippet: Genome-wide identification of transcripts stabilized by TNF in RA FLS. Two biologic replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1 or 72h. Subsequently, Act D was added for 3h and gene expression was measured by RNA sequencing. The degree of TNF-induced mRNA stabilization was calculated as the log 2 difference of TNF+Act D/TNF ratio between 1 and 72h of TNF stimulation and the adjusted p values of TNF-induced stabilization were calculated by RiboDiff. (a), Scatter-plot of the genes displaying TNF-induced mRNA stabilization comparing the degree of mRNA stabilization (y axis) to the adjusted p values of the stabilizing effect of TNF (x-axis). (b), The top 40 genes displaying the highest TNF-induced mRNA stabilization ranked by the degree of stabilization. (c), Enriched biological processes identified by GSEA/MSigDB pathway analysis of the top 10% of the genes (n = 593) displaying the highest degree of TNF-induced mRNA stabilization.

    Article Snippet: RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts.

    Techniques: Genome Wide, Derivative Assay, Activated Clotting Time Assay, Expressing, RNA Sequencing Assay

    TNF induces expression of mRNA-stabilizing pathways and mRNA stabilization is MAPK-dependent. (a), RNA sequencing was performed in 2 biological replicates (derived from two different RA patients) of TNF-stimulated RA FLS and Panther-Gene Ontology was used to evaluate their enrichment for the biological process “Regulation of RNA stability” (GO:0043487 or GO:0043488). F.E = fold enrichment and ns = not significant. (b-h), RA FLS were exposed to a single dose of TNF (10 ng/ml) for 72h and then Act D (10 μg/ml) was added for 20 mins to block active transcription. Subsequently, the cells were treated for 4h with SB202190 (p38 inhibitor) alone or in various combinations with U0126 (MEK inhibitor) and SP600125 (JNK inhibitor). qPCR was used to measure the mRNA levels of CCL5 (b), IL-6 (c), IL-8 (d), CXCL3 (e), CCL2 (f), PTGS2 (g), and CXCL1 (h). Cumulative results from 4 independent experiments are shown. Values were normalized relative to GAPDH mRNA and presented as mean ±SEM. The mRNA expression at the TNF+Act D condition was set to 100 and the mRNA expression at all the other conditions was calculated as % of the TNF+Act D condition. P values were calculated by one-way ANOVA and Tukey post-test analysis (* = p

    Journal: PLoS ONE

    Article Title: Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1371/journal.pone.0179762

    Figure Lengend Snippet: TNF induces expression of mRNA-stabilizing pathways and mRNA stabilization is MAPK-dependent. (a), RNA sequencing was performed in 2 biological replicates (derived from two different RA patients) of TNF-stimulated RA FLS and Panther-Gene Ontology was used to evaluate their enrichment for the biological process “Regulation of RNA stability” (GO:0043487 or GO:0043488). F.E = fold enrichment and ns = not significant. (b-h), RA FLS were exposed to a single dose of TNF (10 ng/ml) for 72h and then Act D (10 μg/ml) was added for 20 mins to block active transcription. Subsequently, the cells were treated for 4h with SB202190 (p38 inhibitor) alone or in various combinations with U0126 (MEK inhibitor) and SP600125 (JNK inhibitor). qPCR was used to measure the mRNA levels of CCL5 (b), IL-6 (c), IL-8 (d), CXCL3 (e), CCL2 (f), PTGS2 (g), and CXCL1 (h). Cumulative results from 4 independent experiments are shown. Values were normalized relative to GAPDH mRNA and presented as mean ±SEM. The mRNA expression at the TNF+Act D condition was set to 100 and the mRNA expression at all the other conditions was calculated as % of the TNF+Act D condition. P values were calculated by one-way ANOVA and Tukey post-test analysis (* = p

    Article Snippet: RNA-sequencing libraries were generated from two patient donor FLS using Tru-Seq kits (Illumina) for poly-A selected transcripts.

    Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Activated Clotting Time Assay, Blocking Assay, Real-time Polymerase Chain Reaction

    Expression level of genes related to alternative geranylgeranyl diphosphate (GGPP) catabolism during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of genes related to alternative geranylgeranyl diphosphate (GGPP) catabolism during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    Expression level of putative ripening transcriptional regulator genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of putative ripening transcriptional regulator genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    Expression level of carotenoid catabolism genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of carotenoid catabolism genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    Expression level of mevalonic acid (MVA) pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of mevalonic acid (MVA) pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    Expression level of 2- C -methyl- d -erythritol-4-phosphate (MEP) pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of 2- C -methyl- d -erythritol-4-phosphate (MEP) pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    Expression level of carotenoid metabolism pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Journal: BMC Genomics

    Article Title: Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    doi: 10.1186/1471-2164-14-781

    Figure Lengend Snippet: Expression level of carotenoid metabolism pathway genes during watermelon ripening. Data were obtained by Illumina RNA Sequencing using two biological replicas and are expressed as Reads Per Kilobase of exon model per Million mapped reads (RPKM). The figure includes only the genes differentially expressed during watermelon fruit ripening with a FDR (False Discovery Rate) ≤ 0.05.

    Article Snippet: The expression profiles of 19,324 genes, among the 23,440 predicted in the watermelon genome assembly [ , ], were generated from trascriptome characterization of watermelon ripening fruits of the Dumara cultivar by Illumina RNA sequencing.

    Techniques: Expressing, RNA Sequencing Assay

    B. burgdorferi in vivo expression technology (BbIVET)-identified sequences with associated TSSs. ( A ) Schematic representation of BbIVET associations with 5′RNA-seq TSSs. Brackets designate the parameters for the association. Relative orientation of the genome region (wide black bar with white arrows), Bbive sequence (thin black arrow) and TSS (orange bent arrow) are indicated. The minimum and maximum Bbive -TSS association distances were defined as 20 nts from the 5′ end and 6 nts from the 3′ end of a Bbive sequence. ( B ) Categorization of Bbive s that associate with 5′RNA-seq TSSs.

    Journal: Nucleic Acids Research

    Article Title: In vivo expression technology and 5′ end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection

    doi: 10.1093/nar/gkw1180

    Figure Lengend Snippet: B. burgdorferi in vivo expression technology (BbIVET)-identified sequences with associated TSSs. ( A ) Schematic representation of BbIVET associations with 5′RNA-seq TSSs. Brackets designate the parameters for the association. Relative orientation of the genome region (wide black bar with white arrows), Bbive sequence (thin black arrow) and TSS (orange bent arrow) are indicated. The minimum and maximum Bbive -TSS association distances were defined as 20 nts from the 5′ end and 6 nts from the 3′ end of a Bbive sequence. ( B ) Categorization of Bbive s that associate with 5′RNA-seq TSSs.

    Article Snippet: This is based on the notion that TAP hydrolyses phosphodiester bonds on RNA 5′ ends and that RNA ligase has specificity to only ligate the 5′ Illumina adapter to RNA sequences harboring a 5′ monophosphate, thereby enriching TSSs to TAP+ libraries.

    Techniques: In Vivo, Expressing, RNA Sequencing Assay, Sequencing

    Novel B. burgdorferi RNA transcripts expressed during murine infection. ( A ) A subset of Bbives that lack or have low read count associated TSSs demonstrate low promoter activity in culture. Bbive sequences were fused to luciferase in pJSB161 and measured for luciferase activity, as described in Figure 3 legend. Data represent the mean ± SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 using the two-tailed Student's t -test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161, ( P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. ( B ) Bioluminescence of Bbive sequences in the murine host. C3H/HeN mice infected intraperitoneally with 1 × 10 5 B. burgdorferi containing controls and specific Bbive luciferase fusions, analyzed by IVIS as described in Figure 4 legend. Symbols indicate specific controls for each time point. Data are representative of two biological replicates. Note that the same color spectrum scale is used between figures.

    Journal: Nucleic Acids Research

    Article Title: In vivo expression technology and 5′ end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection

    doi: 10.1093/nar/gkw1180

    Figure Lengend Snippet: Novel B. burgdorferi RNA transcripts expressed during murine infection. ( A ) A subset of Bbives that lack or have low read count associated TSSs demonstrate low promoter activity in culture. Bbive sequences were fused to luciferase in pJSB161 and measured for luciferase activity, as described in Figure 3 legend. Data represent the mean ± SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 using the two-tailed Student's t -test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161, ( P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. ( B ) Bioluminescence of Bbive sequences in the murine host. C3H/HeN mice infected intraperitoneally with 1 × 10 5 B. burgdorferi containing controls and specific Bbive luciferase fusions, analyzed by IVIS as described in Figure 4 legend. Symbols indicate specific controls for each time point. Data are representative of two biological replicates. Note that the same color spectrum scale is used between figures.

    Article Snippet: This is based on the notion that TAP hydrolyses phosphodiester bonds on RNA 5′ ends and that RNA ligase has specificity to only ligate the 5′ Illumina adapter to RNA sequences harboring a 5′ monophosphate, thereby enriching TSSs to TAP+ libraries.

    Techniques: Infection, Activity Assay, Luciferase, Two Tailed Test, Construct, Mouse Assay

    Genome-wide identification and characterization of B. burgdorferi transcription start sites (TSSs). RNA was isolated from log phase B. burgdorferi clone B31 A3, and treated with and without TAP (tobacco acid phosphatase). ( A ) Schematic classification of the 6042 TSSs relative to the genome organization. Circled numbers indicate TSSs for each category. The maximum nucleotide distances from the 5′ and/or 3′ of annotated sequences, shown as black arrows, for a particular category of TSS are indicated in red. ( B ) Graphical representation of the TSS classifications. as, antisense; o, orphan; p, primary; s, secondary; i, internal. ( C ) Nucleotide frequency at the −100, −1, +1 (TSS) and −1 nucleotide positions. A, adenine, T, thymine, G, guanine, C, cytosine. ( D ) Consensus motif for promoter regions upstream of primary TSSs. The nucleotide sequences from −40 to +1 of the 321 identified primary TSSs were analyzed by MEME 4.11.2. ( E ) Distribution of 5′ untranslated regions (UTR) lengths among uniquely classified primary and secondary TSSs. The data are shown as the percent of mRNA sequences with a 5′ UTR of each bin length. 0–10 nucleotides (red bar), bin size of 20 nucleotides, where the number shown is the middle length in each bin (black bars). ( F ) Consensus ribosome binding motif for uniquely classified, primary 5′ UTRs. MEME 4.11.2 was used to analyze UTR sequences ranging from 10–293 nts in length for a conserved motif, which was found on average to begin 8 nts upstream of the annotated start codon. ( G ) Northern blot analyses validate a long 5′ UTR. Total RNA was extracted from mid-log phase spirochetes and separated by denaturing formaldehyde–agarose gel, blotted to nylon membranes and probed with 32 P-labeled complementary probes, indicated by red and black boxes. Genomic context of ORFs BB_0240-BB_0243 (wide black arrows), the primary TSS (green bent arrow), putative transcripts and their position on the Northern blot (broken line, green arrows and marked with designated symbol) are indicated. Marker sizes in nucleotides are indicated to the left of each blot.

    Journal: Nucleic Acids Research

    Article Title: In vivo expression technology and 5′ end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection

    doi: 10.1093/nar/gkw1180

    Figure Lengend Snippet: Genome-wide identification and characterization of B. burgdorferi transcription start sites (TSSs). RNA was isolated from log phase B. burgdorferi clone B31 A3, and treated with and without TAP (tobacco acid phosphatase). ( A ) Schematic classification of the 6042 TSSs relative to the genome organization. Circled numbers indicate TSSs for each category. The maximum nucleotide distances from the 5′ and/or 3′ of annotated sequences, shown as black arrows, for a particular category of TSS are indicated in red. ( B ) Graphical representation of the TSS classifications. as, antisense; o, orphan; p, primary; s, secondary; i, internal. ( C ) Nucleotide frequency at the −100, −1, +1 (TSS) and −1 nucleotide positions. A, adenine, T, thymine, G, guanine, C, cytosine. ( D ) Consensus motif for promoter regions upstream of primary TSSs. The nucleotide sequences from −40 to +1 of the 321 identified primary TSSs were analyzed by MEME 4.11.2. ( E ) Distribution of 5′ untranslated regions (UTR) lengths among uniquely classified primary and secondary TSSs. The data are shown as the percent of mRNA sequences with a 5′ UTR of each bin length. 0–10 nucleotides (red bar), bin size of 20 nucleotides, where the number shown is the middle length in each bin (black bars). ( F ) Consensus ribosome binding motif for uniquely classified, primary 5′ UTRs. MEME 4.11.2 was used to analyze UTR sequences ranging from 10–293 nts in length for a conserved motif, which was found on average to begin 8 nts upstream of the annotated start codon. ( G ) Northern blot analyses validate a long 5′ UTR. Total RNA was extracted from mid-log phase spirochetes and separated by denaturing formaldehyde–agarose gel, blotted to nylon membranes and probed with 32 P-labeled complementary probes, indicated by red and black boxes. Genomic context of ORFs BB_0240-BB_0243 (wide black arrows), the primary TSS (green bent arrow), putative transcripts and their position on the Northern blot (broken line, green arrows and marked with designated symbol) are indicated. Marker sizes in nucleotides are indicated to the left of each blot.

    Article Snippet: This is based on the notion that TAP hydrolyses phosphodiester bonds on RNA 5′ ends and that RNA ligase has specificity to only ligate the 5′ Illumina adapter to RNA sequences harboring a 5′ monophosphate, thereby enriching TSSs to TAP+ libraries.

    Techniques: Genome Wide, Isolation, Binding Assay, Northern Blot, Agarose Gel Electrophoresis, Labeling, Marker

    Validation of BbIVET-associated RNA transcripts. ( A ) Deep-sequencing screen shot for a Bbive -internal TSS, displaying only the sequenced 5′ nucleotide, of overlaid biological replicates treated with (TAP+) and without (TAP−) tobacco acid pyrophosphatase. Read count ranges are shown in the upper left of each frame. The chromosome nucleotide coordinates, relative orientation of the BB_0370 ORF (wide black bar), Bbive45 sequence (thin black arrow), internal TSS (blue bent arrow), processed 5′ ends (scissors), putative transcripts (broken line arrows), Northern probe locations (black and red boxes) and luciferase fusion regions (brackets) are indicated. The predicted transcripts of interest are marked with an asterisk. ( B ) Northern blot analyses of the Bbive45 internal TSS, as described in the Figure 1 legend. Probes located upstream (red) and downstream (black) of the putative internal TSS, are indicated. Marker nucleotide sizes are indicated to the left of the blots. ( C ) Bbive45 promoter activity and specificity. B. burgdorferi clones harboring specific promoter fusions were grown to mid-log phase, and incubated with 750 μM D-luciferin. Relative luciferase units (RLU) were normalized to cell density by OD 600 . Data represent the mean ±SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 with the two-tailed Student's t-test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161 ( P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. ( D ) Schematic representation of Bbive luciferase fusions. The bracket designates the sequence selected for promoter fusion to luciferase in pJSB161. Relative orientation of the genome region (wide black bar with white arrows), 5′ boundary of Bbive sequence (orange line) and TSS (orange bent arrow) are indicated. ( E ) A variety of Bbives with associated TSSs have promoter activity in culture. Spirochetes containing control promoters ( flaBp, ospCp and ospAp ) and specific Bbives , fused to luciferase, were grown to mid-log phase, and analyzed as described above.

    Journal: Nucleic Acids Research

    Article Title: In vivo expression technology and 5′ end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection

    doi: 10.1093/nar/gkw1180

    Figure Lengend Snippet: Validation of BbIVET-associated RNA transcripts. ( A ) Deep-sequencing screen shot for a Bbive -internal TSS, displaying only the sequenced 5′ nucleotide, of overlaid biological replicates treated with (TAP+) and without (TAP−) tobacco acid pyrophosphatase. Read count ranges are shown in the upper left of each frame. The chromosome nucleotide coordinates, relative orientation of the BB_0370 ORF (wide black bar), Bbive45 sequence (thin black arrow), internal TSS (blue bent arrow), processed 5′ ends (scissors), putative transcripts (broken line arrows), Northern probe locations (black and red boxes) and luciferase fusion regions (brackets) are indicated. The predicted transcripts of interest are marked with an asterisk. ( B ) Northern blot analyses of the Bbive45 internal TSS, as described in the Figure 1 legend. Probes located upstream (red) and downstream (black) of the putative internal TSS, are indicated. Marker nucleotide sizes are indicated to the left of the blots. ( C ) Bbive45 promoter activity and specificity. B. burgdorferi clones harboring specific promoter fusions were grown to mid-log phase, and incubated with 750 μM D-luciferin. Relative luciferase units (RLU) were normalized to cell density by OD 600 . Data represent the mean ±SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 with the two-tailed Student's t-test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161 ( P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. ( D ) Schematic representation of Bbive luciferase fusions. The bracket designates the sequence selected for promoter fusion to luciferase in pJSB161. Relative orientation of the genome region (wide black bar with white arrows), 5′ boundary of Bbive sequence (orange line) and TSS (orange bent arrow) are indicated. ( E ) A variety of Bbives with associated TSSs have promoter activity in culture. Spirochetes containing control promoters ( flaBp, ospCp and ospAp ) and specific Bbives , fused to luciferase, were grown to mid-log phase, and analyzed as described above.

    Article Snippet: This is based on the notion that TAP hydrolyses phosphodiester bonds on RNA 5′ ends and that RNA ligase has specificity to only ligate the 5′ Illumina adapter to RNA sequences harboring a 5′ monophosphate, thereby enriching TSSs to TAP+ libraries.

    Techniques: Sequencing, Northern Blot, Luciferase, Marker, Activity Assay, Clone Assay, Incubation, Two Tailed Test, Construct

    The schematic diagram shows sample processing of human Sertoli cells from OA patients and NOA patients, cell pooling, and RNA-Seq data processing procedure

    Journal: Stem Cell Research & Therapy

    Article Title: Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation

    doi: 10.1186/s13287-019-1139-7

    Figure Lengend Snippet: The schematic diagram shows sample processing of human Sertoli cells from OA patients and NOA patients, cell pooling, and RNA-Seq data processing procedure

    Article Snippet: RNA isolation, library construction, and Illumina sequencing Total RNA was extracted from human Sertoli cells using RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) to remove genomic DNA.

    Techniques: RNA Sequencing Assay

    Expression of FGF5 in the human testis of OA and SCOS patients. a RT-PCR showed the expression of FGF5 , FGFR1 , and FGFR2 in freshly isolated human Sertoli cells. ACTB was used as a loading control of total RNA. b Immunocytochemistry showed FGF5 expression in human Sertoli cells of OA and SCOS patients. c Western blot confirmed that expression of FGF5 was decreased at protein level in Sertoli cells from SCOS patients. d IHC revealed the location of FGF5 expression in the human testis (scale bar = 10 μm). Asterisk indicated the location of FGF5 expression in the human testicular section

    Journal: Stem Cell Research & Therapy

    Article Title: Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation

    doi: 10.1186/s13287-019-1139-7

    Figure Lengend Snippet: Expression of FGF5 in the human testis of OA and SCOS patients. a RT-PCR showed the expression of FGF5 , FGFR1 , and FGFR2 in freshly isolated human Sertoli cells. ACTB was used as a loading control of total RNA. b Immunocytochemistry showed FGF5 expression in human Sertoli cells of OA and SCOS patients. c Western blot confirmed that expression of FGF5 was decreased at protein level in Sertoli cells from SCOS patients. d IHC revealed the location of FGF5 expression in the human testis (scale bar = 10 μm). Asterisk indicated the location of FGF5 expression in the human testicular section

    Article Snippet: RNA isolation, library construction, and Illumina sequencing Total RNA was extracted from human Sertoli cells using RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) to remove genomic DNA.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Immunocytochemistry, Western Blot, Immunohistochemistry

    Morphology of the testis from OA and SCOS patients and identifications of isolated human Sertoli cells. a H E staining illustrated the morphology of testicular tissues from OA (left panel) and SCOS patients (right panel) (scale bar = 20 μm). b RT-PCR showed that the human Sertoli cells isolated from OA (1) and SCOS (2) patients’ testes expressed transcripts of GATA4 , ABP , WT1 , and FSHR . ACTB (actin beta) was used as a loading control of total RNA. c Immunohistochemistry reveals that GATA4 is expressed in human Sertoli cells isolated from OA and SCOS patients’ testes (scale bar = 10 μm). Note: OA obstructive azoospermia, SCOS Sertoli cell-only syndrome, SC Sertoli cell

    Journal: Stem Cell Research & Therapy

    Article Title: Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation

    doi: 10.1186/s13287-019-1139-7

    Figure Lengend Snippet: Morphology of the testis from OA and SCOS patients and identifications of isolated human Sertoli cells. a H E staining illustrated the morphology of testicular tissues from OA (left panel) and SCOS patients (right panel) (scale bar = 20 μm). b RT-PCR showed that the human Sertoli cells isolated from OA (1) and SCOS (2) patients’ testes expressed transcripts of GATA4 , ABP , WT1 , and FSHR . ACTB (actin beta) was used as a loading control of total RNA. c Immunohistochemistry reveals that GATA4 is expressed in human Sertoli cells isolated from OA and SCOS patients’ testes (scale bar = 10 μm). Note: OA obstructive azoospermia, SCOS Sertoli cell-only syndrome, SC Sertoli cell

    Article Snippet: RNA isolation, library construction, and Illumina sequencing Total RNA was extracted from human Sertoli cells using RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) to remove genomic DNA.

    Techniques: Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

    Differentially expressed genes in RNA-Seq were confirmed by Q-PCR. A total of 20 genes, including 10 SCOS Sertoli cells downregulated ( FGF5 , DKK1 , IGF2 , PNP , SEMA3A , CDH1 , SPRY2 , CDK6 , MET and WNK4 ) ( a ) and upregulated ( b ) ones ( SPP1 , TP53I11 , ADRA2A , PLA2G2A , NTN1 , OSCAR , PRRX2 , WFDC1 , NGFR and MT3 ), from 308 DEGs were selected to validate the results of RNA-Seq. Note: OA obstructive azoospermia, SCOS Sertoli cell-only syndrome, SC Sertoli cell. Asterisk indicated statistically significant differences ( P

    Journal: Stem Cell Research & Therapy

    Article Title: Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation

    doi: 10.1186/s13287-019-1139-7

    Figure Lengend Snippet: Differentially expressed genes in RNA-Seq were confirmed by Q-PCR. A total of 20 genes, including 10 SCOS Sertoli cells downregulated ( FGF5 , DKK1 , IGF2 , PNP , SEMA3A , CDH1 , SPRY2 , CDK6 , MET and WNK4 ) ( a ) and upregulated ( b ) ones ( SPP1 , TP53I11 , ADRA2A , PLA2G2A , NTN1 , OSCAR , PRRX2 , WFDC1 , NGFR and MT3 ), from 308 DEGs were selected to validate the results of RNA-Seq. Note: OA obstructive azoospermia, SCOS Sertoli cell-only syndrome, SC Sertoli cell. Asterisk indicated statistically significant differences ( P

    Article Snippet: RNA isolation, library construction, and Illumina sequencing Total RNA was extracted from human Sertoli cells using RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) to remove genomic DNA.

    Techniques: RNA Sequencing Assay, Polymerase Chain Reaction

    Reannotation of the lytic VZV transcriptome reveals novel viral transcript isoforms, fusion transcripts and putative noncoding RNAs. The reannotated lytic VZV transcriptome includes 71 transcripts encoding canonical ORFs with their UTRs defined (grey), 39 alternative isoforms of existing RNAs (orange), 7 fusion RNAs (dark blue) and 19 novel polyadenylated RNAs (red). Wide and thin boxes indicate canonical CDS domains and while thin boxes indicate UTRs, respectively. Absence of CDS regions indicates that the respective VZV RNA has an uncertain coding potential. Illumina RNA-Seq (light blue) and nanopore dRNA-Seq (teal) coverage plots are derived from ARPE-19 cells lytically infected with VZV strain pOka for 96 hr. Y-axis values indicate the maximum read depth of that track. See also Figures S1 and S2 . Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: Reannotation of the lytic VZV transcriptome reveals novel viral transcript isoforms, fusion transcripts and putative noncoding RNAs. The reannotated lytic VZV transcriptome includes 71 transcripts encoding canonical ORFs with their UTRs defined (grey), 39 alternative isoforms of existing RNAs (orange), 7 fusion RNAs (dark blue) and 19 novel polyadenylated RNAs (red). Wide and thin boxes indicate canonical CDS domains and while thin boxes indicate UTRs, respectively. Absence of CDS regions indicates that the respective VZV RNA has an uncertain coding potential. Illumina RNA-Seq (light blue) and nanopore dRNA-Seq (teal) coverage plots are derived from ARPE-19 cells lytically infected with VZV strain pOka for 96 hr. Y-axis values indicate the maximum read depth of that track. See also Figures S1 and S2 . Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: RNA Sequencing Assay, Derivative Assay, Infection

    VZV transcriptome diversity is consistent between viral strains. Coverage plots derived from Nanopore dRNA-Seq of VZV strain pOka (blue) and VZV strain EMC-1 (red)-infected ARPE-19 cells at 96 hpi. Note that while relative and absolute abundances of distinct VZV RNAs differ between strains, all RNAs included in our annotation are represented. Y-axis denote coverage range. Canonical (grey), alternative (orange), fusion (dark blue) and putative ncRNAs (red) are shown with canonical CDS regions indicated by wide boxes and UTRs shown as thin boxes. The absence of a CDS region indicates that an RNA has uncertain coding potential. The reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. The scatter plot shows the correlation between VZV transcript abundance in VZV EMC-1-infected and VZV pOka-infected ARPE-19 cells. Pearson R 2 and p -value are indicated.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: VZV transcriptome diversity is consistent between viral strains. Coverage plots derived from Nanopore dRNA-Seq of VZV strain pOka (blue) and VZV strain EMC-1 (red)-infected ARPE-19 cells at 96 hpi. Note that while relative and absolute abundances of distinct VZV RNAs differ between strains, all RNAs included in our annotation are represented. Y-axis denote coverage range. Canonical (grey), alternative (orange), fusion (dark blue) and putative ncRNAs (red) are shown with canonical CDS regions indicated by wide boxes and UTRs shown as thin boxes. The absence of a CDS region indicates that an RNA has uncertain coding potential. The reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. The scatter plot shows the correlation between VZV transcript abundance in VZV EMC-1-infected and VZV pOka-infected ARPE-19 cells. Pearson R 2 and p -value are indicated.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: Derivative Assay, Infection

    Expression of ORF0 RNA isoforms during lytic infection of ARPE-19 cells with VZV pOka and EMC-1. Identical TSS and CPAS sites were detected in ARPE-19 cells infected with cell-free VZV pOka (VZV clade 2; yellow) or -EMC-1 (VZV clade 1; green) at 96 hpi, yielding two ORF0 RNA isoforms. However, due to nucleotide variability between both strains, VZV pOka RNA 0-1 includes a larger CDS and can encode an alternative ORF0 protein variant, compared to VZV EMC-1. Nanopore dRNA-Seq (teal) coverage plots are shown. Y-axis values indicate the maximum read depth of that specific track. CDS regions and UTRs are shown as wide and thin boxes, respectively.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: Expression of ORF0 RNA isoforms during lytic infection of ARPE-19 cells with VZV pOka and EMC-1. Identical TSS and CPAS sites were detected in ARPE-19 cells infected with cell-free VZV pOka (VZV clade 2; yellow) or -EMC-1 (VZV clade 1; green) at 96 hpi, yielding two ORF0 RNA isoforms. However, due to nucleotide variability between both strains, VZV pOka RNA 0-1 includes a larger CDS and can encode an alternative ORF0 protein variant, compared to VZV EMC-1. Nanopore dRNA-Seq (teal) coverage plots are shown. Y-axis values indicate the maximum read depth of that specific track. CDS regions and UTRs are shown as wide and thin boxes, respectively.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: Expressing, Infection, Variant Assay

    Decoding the complexity of the lytic VZV transcriptome. (A) Experimental strategy: ARPE-19 cells and hESC-derived neurons were infected with VZV EMC-1 (Clade 1) or -pOka (Clade 2) for 96 hrs. Total RNA was extracted and poly(A) fraction isolated for sequencing by Illumina CAGE-Seq, Illumina RNA-Seq and/or Nanopore dRNA-Seq. Sequence data were aligned against the VZV strain Dumas reference genome (Genbank Accession: NC_001348.1 ) and visualized using the Integrative Genomics Viewer (IGV) ( Thorvaldsdóttir et al., 2013 ) and GVIZ ( Hahne and Ivanek, 2016 ). (B) Transcription start sites (TSS) as well as cleavage and polyadenylation sites (CPAS) were identified in nanopore and Illumina datasets. Histograms show the distances observed between nanopore and Illumina predictions, while inset Venn diagrams indicate the numbers of sites identified and their conservation between datasets. (C) Integration of Illumina RNA-Seq, Illumina CAGE-Seq and Nanopore dRNA-Seq datasets. Coverage plots for Illumina RNA-Seq (light-blue), CAGE-Seq (red) and Nanopore dRNA-Seq (teal) are integrated with pileup data that maps TSS (red) and CPAS (black). Rows denoted by TSS and CPAS indicate positions of TSS and CPAS identified using HOMER software ( Heinz et al., 2010 ) for nanopore dRNA-Seq and Illumina CAGE-Seq data and ContextMap2 software ( Bonfert et al., 2015 ) for Illumina RNA-Seq data. Conserved TSS and CPAS are indicated with blue asterisks. RNA structures (grey) are inferred from these conserved sites. Wide and thin boxes indicate canonical coding sequence (CDS) domains and untranslated regions (UTRs), respectively. Novel identified RNAs (orange) are shown without predicted CDS domains.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: Decoding the complexity of the lytic VZV transcriptome. (A) Experimental strategy: ARPE-19 cells and hESC-derived neurons were infected with VZV EMC-1 (Clade 1) or -pOka (Clade 2) for 96 hrs. Total RNA was extracted and poly(A) fraction isolated for sequencing by Illumina CAGE-Seq, Illumina RNA-Seq and/or Nanopore dRNA-Seq. Sequence data were aligned against the VZV strain Dumas reference genome (Genbank Accession: NC_001348.1 ) and visualized using the Integrative Genomics Viewer (IGV) ( Thorvaldsdóttir et al., 2013 ) and GVIZ ( Hahne and Ivanek, 2016 ). (B) Transcription start sites (TSS) as well as cleavage and polyadenylation sites (CPAS) were identified in nanopore and Illumina datasets. Histograms show the distances observed between nanopore and Illumina predictions, while inset Venn diagrams indicate the numbers of sites identified and their conservation between datasets. (C) Integration of Illumina RNA-Seq, Illumina CAGE-Seq and Nanopore dRNA-Seq datasets. Coverage plots for Illumina RNA-Seq (light-blue), CAGE-Seq (red) and Nanopore dRNA-Seq (teal) are integrated with pileup data that maps TSS (red) and CPAS (black). Rows denoted by TSS and CPAS indicate positions of TSS and CPAS identified using HOMER software ( Heinz et al., 2010 ) for nanopore dRNA-Seq and Illumina CAGE-Seq data and ContextMap2 software ( Bonfert et al., 2015 ) for Illumina RNA-Seq data. Conserved TSS and CPAS are indicated with blue asterisks. RNA structures (grey) are inferred from these conserved sites. Wide and thin boxes indicate canonical coding sequence (CDS) domains and untranslated regions (UTRs), respectively. Novel identified RNAs (orange) are shown without predicted CDS domains.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: Derivative Assay, Infection, Isolation, Sequencing, RNA Sequencing Assay, Software

    VZV transcriptome diversity is consistent between infected cell types. Coverage plots derived from Nanopore dRNA-Seq of VZV pOka infected ARPE-19 cells (blue) and hESC-derived neurons (purple). RNA was collected at 96 hpi (ARPE-19 cells) or 144 hpi (neurons). Note that while relative and absolute abundances of distinct VZV RNAs differ between cell types, all RNAs included in our annotation are represented. Y-axis denote coverage range. Canonical (grey), alternative (orange), fusion (dark blue) and putative ncRNAs (red) are shown with canonical CDS regions indicated by wide boxes and UTRs shown as thin boxes. The absence of CDS region indicates that the respective VZV RNA has an uncertain coding potential. Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. The scatter plot shows the correlation between VZV transcript abundance in hESC-derived neurons and ARPE-19 cells. Pearson R2 and p-value are indicated.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: VZV transcriptome diversity is consistent between infected cell types. Coverage plots derived from Nanopore dRNA-Seq of VZV pOka infected ARPE-19 cells (blue) and hESC-derived neurons (purple). RNA was collected at 96 hpi (ARPE-19 cells) or 144 hpi (neurons). Note that while relative and absolute abundances of distinct VZV RNAs differ between cell types, all RNAs included in our annotation are represented. Y-axis denote coverage range. Canonical (grey), alternative (orange), fusion (dark blue) and putative ncRNAs (red) are shown with canonical CDS regions indicated by wide boxes and UTRs shown as thin boxes. The absence of CDS region indicates that the respective VZV RNA has an uncertain coding potential. Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. The scatter plot shows the correlation between VZV transcript abundance in hESC-derived neurons and ARPE-19 cells. Pearson R2 and p-value are indicated.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: Infection, Derivative Assay

    Kinetic class of viral RNAs expressed during lytic VZV infection of ARPE-19 cells. (A) Coverage plots derived from Illumina RNA-Seq of ARPE-19 cells infected with cell-free VZV EMC-1 and treated with actinomycin D (ActD; grey), cycloheximide (CHX; green), phosphonoacetic acid (PAA; gold), or untreated (UNT; red). Y-axis denotes coverage range. All transcripts are colour-coded according to their assigned kinetic class: IE – green, E – gold, LL – red, TL – dark red. Canonical CDS regions are indicated by wide boxes with UTRs shown as thin boxes. Absence of CDS region indicates that an RNA has an uncertain coding potential. Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. ( B ) The inset black hatched box presents a close-up view of the RNA 9 locus.

    Journal: bioRxiv

    Article Title: Decoding the architecture of the varicella-zoster virus transcriptome

    doi: 10.1101/2020.05.25.110965

    Figure Lengend Snippet: Kinetic class of viral RNAs expressed during lytic VZV infection of ARPE-19 cells. (A) Coverage plots derived from Illumina RNA-Seq of ARPE-19 cells infected with cell-free VZV EMC-1 and treated with actinomycin D (ActD; grey), cycloheximide (CHX; green), phosphonoacetic acid (PAA; gold), or untreated (UNT; red). Y-axis denotes coverage range. All transcripts are colour-coded according to their assigned kinetic class: IE – green, E – gold, LL – red, TL – dark red. Canonical CDS regions are indicated by wide boxes with UTRs shown as thin boxes. Absence of CDS region indicates that an RNA has an uncertain coding potential. Reiterative repeat regions R1-R5 and both copies of the OriS are shown as black boxes embedded in the genome track. ( B ) The inset black hatched box presents a close-up view of the RNA 9 locus.

    Article Snippet: Decoding the complexity of lytic VZV gene expression Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures.

    Techniques: Infection, Derivative Assay, RNA Sequencing Assay

    (a) Haplotype network of the combined three cp DNA ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F); (b) Haplotype network of the combined four DNA ( ITS 2, psb A‐ trn H, mat K, and rbc L) regions using median joining method. Blue dots stand for Tripterygium wilfordii , red dots stand for Tripterygium hypoglaucum , green dots stands for Tripterygium regelii , and black dots stand for Celastrus orbiculatus

    Journal: Ecology and Evolution

    Article Title: Phylogeographic and phylogenetic analysis for Tripterygium species delimitation, et al. Phylogeographic and phylogenetic analysis for Tripterygium species delimitation

    doi: 10.1002/ece3.3344

    Figure Lengend Snippet: (a) Haplotype network of the combined three cp DNA ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F); (b) Haplotype network of the combined four DNA ( ITS 2, psb A‐ trn H, mat K, and rbc L) regions using median joining method. Blue dots stand for Tripterygium wilfordii , red dots stand for Tripterygium hypoglaucum , green dots stands for Tripterygium regelii , and black dots stand for Celastrus orbiculatus

    Article Snippet: The other four DNA sequences (i.e., ITS2, psb A‐trn H, mat K, and rbc L) were downloaded from the National Center for Biotechnology Information (NCBI) nucleotide database ( https://www.ncbi.nlm.nih.gov/nuccore ) and combined as the second matrix, which was used in the phylogenetic analysis, including Bayesian inference (BI), maximum likelihood (ML), and haplotype network construction.

    Techniques:

    BEAST ‐derived chronogram of haplotypes based on the combined cp DNA ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F) sequences. Blue bars on each node show 95% highest posterior density ( HPD ) confidence intervals for divergence estimates. The number on each branch indicates the posterior probability ( PP ). The node age (Myr) of the major lineages was shown near the blue bars

    Journal: Ecology and Evolution

    Article Title: Phylogeographic and phylogenetic analysis for Tripterygium species delimitation, et al. Phylogeographic and phylogenetic analysis for Tripterygium species delimitation

    doi: 10.1002/ece3.3344

    Figure Lengend Snippet: BEAST ‐derived chronogram of haplotypes based on the combined cp DNA ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F) sequences. Blue bars on each node show 95% highest posterior density ( HPD ) confidence intervals for divergence estimates. The number on each branch indicates the posterior probability ( PP ). The node age (Myr) of the major lineages was shown near the blue bars

    Article Snippet: The other four DNA sequences (i.e., ITS2, psb A‐trn H, mat K, and rbc L) were downloaded from the National Center for Biotechnology Information (NCBI) nucleotide database ( https://www.ncbi.nlm.nih.gov/nuccore ) and combined as the second matrix, which was used in the phylogenetic analysis, including Bayesian inference (BI), maximum likelihood (ML), and haplotype network construction.

    Techniques: Derivative Assay

    Phylogenetic trees of haplotypes based on the combined three plastid DNA regions (a) ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F) and the combined four DNA regions (b) ( ITS 2, psb A‐ trn H, mat K, and rbc L) using Bayesian inference method. The number on each branch indicates the posterior probability ( PP ). Model selection: (a) GTR +I+G; (b) GTR +I. The average standard deviation of the split frequencies, (a) 0.006417; (b) 0.002667

    Journal: Ecology and Evolution

    Article Title: Phylogeographic and phylogenetic analysis for Tripterygium species delimitation, et al. Phylogeographic and phylogenetic analysis for Tripterygium species delimitation

    doi: 10.1002/ece3.3344

    Figure Lengend Snippet: Phylogenetic trees of haplotypes based on the combined three plastid DNA regions (a) ( psb A‐t rn H, rpl 32‐ trn L, and trn L‐ trn F) and the combined four DNA regions (b) ( ITS 2, psb A‐ trn H, mat K, and rbc L) using Bayesian inference method. The number on each branch indicates the posterior probability ( PP ). Model selection: (a) GTR +I+G; (b) GTR +I. The average standard deviation of the split frequencies, (a) 0.006417; (b) 0.002667

    Article Snippet: The other four DNA sequences (i.e., ITS2, psb A‐trn H, mat K, and rbc L) were downloaded from the National Center for Biotechnology Information (NCBI) nucleotide database ( https://www.ncbi.nlm.nih.gov/nuccore ) and combined as the second matrix, which was used in the phylogenetic analysis, including Bayesian inference (BI), maximum likelihood (ML), and haplotype network construction.

    Techniques: Selection, Standard Deviation

    Titration of the DNA donor and integrase concentrations for the strand transfer assay. (A) Purification of HIV-O recombinant integrase proteins from clades A (IN-O/A) and B (IN-O/B) and a divergent strain (IN-O/Div) and HIV-M recombinant integrase protein (IN-M/B). SDS-PAGE analysis showing that the integrase proteins resolved predominantly in a high-density band (35.6 kDa); the two bands at the bottom represent nonspecifically cleaved forms of the protein. (B) Calibration of the DNA donor mimicking LTR-O using various concentrations, ranging from 0 to 2,400 nM; (C) calibration of the different HIV-O integrases, IN-O/A (clade A), IN-O/B (clade B), and IN-O/Div (divergent strain), and the HIV-M integrase, IN-M/B (HIV-M subtype B), using various concentrations, ranging from 0 to 1,600 nM.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: HIV-1 Group O Integrase Displays Lower Enzymatic Efficiency and Higher Susceptibility to Raltegravir than HIV-1 Group M Subtype B Integrase

    doi: 10.1128/AAC.03819-14

    Figure Lengend Snippet: Titration of the DNA donor and integrase concentrations for the strand transfer assay. (A) Purification of HIV-O recombinant integrase proteins from clades A (IN-O/A) and B (IN-O/B) and a divergent strain (IN-O/Div) and HIV-M recombinant integrase protein (IN-M/B). SDS-PAGE analysis showing that the integrase proteins resolved predominantly in a high-density band (35.6 kDa); the two bands at the bottom represent nonspecifically cleaved forms of the protein. (B) Calibration of the DNA donor mimicking LTR-O using various concentrations, ranging from 0 to 2,400 nM; (C) calibration of the different HIV-O integrases, IN-O/A (clade A), IN-O/B (clade B), and IN-O/Div (divergent strain), and the HIV-M integrase, IN-M/B (HIV-M subtype B), using various concentrations, ranging from 0 to 1,600 nM.

    Article Snippet: DNA sequences were compared with the HIV-1 group M reference strain HxB2 ( ) using BioEdit software for alignment (BioEdit sequence alignment editor v7.1.3, copyright 1997-2011, Tom Hall).

    Techniques: Titration, Purification, Recombinant, SDS Page

    Enzymatic activity of the different HIV-O integrases from clade A (IN-O/A), clade B (IN-O/B), and divergent strain (IN-O/Div) in comparison with HIV-M integrase (IN-M/B). (A) Strand transfer activity: these data were generated using various concentrations of DNA target (0 to 120 nM); (B) 3′ processing activity: the data were generated using various concentrations of LTR-O (0- to 40 nM).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: HIV-1 Group O Integrase Displays Lower Enzymatic Efficiency and Higher Susceptibility to Raltegravir than HIV-1 Group M Subtype B Integrase

    doi: 10.1128/AAC.03819-14

    Figure Lengend Snippet: Enzymatic activity of the different HIV-O integrases from clade A (IN-O/A), clade B (IN-O/B), and divergent strain (IN-O/Div) in comparison with HIV-M integrase (IN-M/B). (A) Strand transfer activity: these data were generated using various concentrations of DNA target (0 to 120 nM); (B) 3′ processing activity: the data were generated using various concentrations of LTR-O (0- to 40 nM).

    Article Snippet: DNA sequences were compared with the HIV-1 group M reference strain HxB2 ( ) using BioEdit software for alignment (BioEdit sequence alignment editor v7.1.3, copyright 1997-2011, Tom Hall).

    Techniques: Activity Assay, Generated

    Aptamer Binding Selectively Reduces Mutant Huntingtin’s PRC2-Stimulating Activity (A) The affinities of Q78-huntingtin alone and huntingtin-aptamer complexes to PRC2 were compared by immunoprecipitation with an anti-EZH2 antibody and an anti-huntingtin antibody (HP-1), followed by immunoblotting for huntingtin and EZH2. The experiment was repeated three times. (B) Autoradiogram of bands of 3 H-methyl histone H3 produced by PRC2 in the absence and presence of mutant-huntingtin-preferring DNA aptamers. Bottom: the bar graph of the band densitometry results, showing that aptamer binding significantly reduced the ability of mutant huntingtin to enhance basal PRC2 activity. Mean band intensities were measured from three independent experiments. Asterisks indicate statistically significant differences compared with unbound Q78-huntingtin. Error bars represent SEM. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity

    doi: 10.1016/j.omtn.2018.03.008

    Figure Lengend Snippet: Aptamer Binding Selectively Reduces Mutant Huntingtin’s PRC2-Stimulating Activity (A) The affinities of Q78-huntingtin alone and huntingtin-aptamer complexes to PRC2 were compared by immunoprecipitation with an anti-EZH2 antibody and an anti-huntingtin antibody (HP-1), followed by immunoblotting for huntingtin and EZH2. The experiment was repeated three times. (B) Autoradiogram of bands of 3 H-methyl histone H3 produced by PRC2 in the absence and presence of mutant-huntingtin-preferring DNA aptamers. Bottom: the bar graph of the band densitometry results, showing that aptamer binding significantly reduced the ability of mutant huntingtin to enhance basal PRC2 activity. Mean band intensities were measured from three independent experiments. Asterisks indicate statistically significant differences compared with unbound Q78-huntingtin. Error bars represent SEM. *p

    Article Snippet: Three lysine aspartic acid (KD) mutant huntingtin constructs (K2449D, K2932D/K2934D, K2449D/K2932D/K2934D) were generated by submission of the DNA sequences to Genscript (Piscataway, NJ, USA), which provided synthesized DNA in the pFastBac1 vector using SalI/SacII restriction digest and standard molecular biology techniques.

    Techniques: Binding Assay, Mutagenesis, Activity Assay, Immunoprecipitation, Produced

    Screening of DNA Aptamers that Can Preferentially Bind to Q78-Huntingtin (A) A schematic illustrating DNA aptamer screening to identify aptamers that bind preferentially to Q23- or Q78-purified human recombinant huntingtin. (B) Bar graphs presenting ELISA data show four representative aptamers that bind significantly more to the purified Q78-huntingtin (Q78) compared with Q23-huntingtin (Q23). Mean optical densities at 405 nm were measured by ELISA plate reader from three independent experiments. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity

    doi: 10.1016/j.omtn.2018.03.008

    Figure Lengend Snippet: Screening of DNA Aptamers that Can Preferentially Bind to Q78-Huntingtin (A) A schematic illustrating DNA aptamer screening to identify aptamers that bind preferentially to Q23- or Q78-purified human recombinant huntingtin. (B) Bar graphs presenting ELISA data show four representative aptamers that bind significantly more to the purified Q78-huntingtin (Q78) compared with Q23-huntingtin (Q23). Mean optical densities at 405 nm were measured by ELISA plate reader from three independent experiments. *p

    Article Snippet: Three lysine aspartic acid (KD) mutant huntingtin constructs (K2449D, K2932D/K2934D, K2449D/K2932D/K2934D) were generated by submission of the DNA sequences to Genscript (Piscataway, NJ, USA), which provided synthesized DNA in the pFastBac1 vector using SalI/SacII restriction digest and standard molecular biology techniques.

    Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay

    CTD-II Domain near K2932/K2934 in Mutant Huntingtin Is a Potential Binding Site of the MS3 Aptamer (A) A schematic view of huntingtin with five domains delineated as previously described. 8 Caspase-6 cleavage site is indicated by the red arrow. The polyglutamine tract is denoted by the green bar. (B) The biotinylated DNA aptamer MS1 (Biotin, first blot) was detected in uncleaved Q78-huntingtin, and the larger fragment from caspase-6 cleavage of Q78-huntingtin with an anti-biotin antibody. The representative blot showed that most of the aptamer appeared to be bound to the C-terminal fragment and un-cleaved huntingtin (arrows), but not the amino-terminal fragment (arrowhead), which was confirmed by re-probing the blot with a huntingtin C-terminal antibody (HF1, second blot) and an amino-terminal antibody (mAb2166, third blot). The experiment was repeated three times. (C) The same experiment was performed for the remaining biotinylated aptamers (MS2, MS3, MS4) and MS1. The representative immunoblot demonstrated that all aptamers preferentially bound to the C terminus of Q78-huntingtin. The experiment was repeated three times. (D and E) SLM analysis was performed with the huntingtin-aptamer complex and huntingtin alone. The methylated lysine residues were detected by LC/MS/MS and assigned a score based on the equation described in the Materials and Methods . The bar graphs showing the score of Q23- (D) and Q78- (E) huntingtin revealed that K2932/K2934 in CTD-II was the most probable binding site of MS3 on Q78-huntingtin. (F) Representative immunoblot showing purified Q23-, Q78-, Q78 K2449D-, Q78 K2932D/K2934D-, and Q78 K2449D/K2932D/K2934D-huntingtin bound with biotinylated MS3 or GCdx aptamers by probing with an anti-biotin antibody (Biotin) and an anti-huntingtin antibody (mAb2166). The location of full-length huntingtin is indicated by the arrow in both immunoblots. The asterisk marked a blurry band detected by anti-biotin antibody, but not by mAb2166, implying that a protein (or more than one) from insect cells, showing affinities to MS3 aptamer, was particularly enriched in Q78 K2449D-purified protein and less in Q78 K2932D/K2934D-purified proteins. The experiment was repeated three times.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity

    doi: 10.1016/j.omtn.2018.03.008

    Figure Lengend Snippet: CTD-II Domain near K2932/K2934 in Mutant Huntingtin Is a Potential Binding Site of the MS3 Aptamer (A) A schematic view of huntingtin with five domains delineated as previously described. 8 Caspase-6 cleavage site is indicated by the red arrow. The polyglutamine tract is denoted by the green bar. (B) The biotinylated DNA aptamer MS1 (Biotin, first blot) was detected in uncleaved Q78-huntingtin, and the larger fragment from caspase-6 cleavage of Q78-huntingtin with an anti-biotin antibody. The representative blot showed that most of the aptamer appeared to be bound to the C-terminal fragment and un-cleaved huntingtin (arrows), but not the amino-terminal fragment (arrowhead), which was confirmed by re-probing the blot with a huntingtin C-terminal antibody (HF1, second blot) and an amino-terminal antibody (mAb2166, third blot). The experiment was repeated three times. (C) The same experiment was performed for the remaining biotinylated aptamers (MS2, MS3, MS4) and MS1. The representative immunoblot demonstrated that all aptamers preferentially bound to the C terminus of Q78-huntingtin. The experiment was repeated three times. (D and E) SLM analysis was performed with the huntingtin-aptamer complex and huntingtin alone. The methylated lysine residues were detected by LC/MS/MS and assigned a score based on the equation described in the Materials and Methods . The bar graphs showing the score of Q23- (D) and Q78- (E) huntingtin revealed that K2932/K2934 in CTD-II was the most probable binding site of MS3 on Q78-huntingtin. (F) Representative immunoblot showing purified Q23-, Q78-, Q78 K2449D-, Q78 K2932D/K2934D-, and Q78 K2449D/K2932D/K2934D-huntingtin bound with biotinylated MS3 or GCdx aptamers by probing with an anti-biotin antibody (Biotin) and an anti-huntingtin antibody (mAb2166). The location of full-length huntingtin is indicated by the arrow in both immunoblots. The asterisk marked a blurry band detected by anti-biotin antibody, but not by mAb2166, implying that a protein (or more than one) from insect cells, showing affinities to MS3 aptamer, was particularly enriched in Q78 K2449D-purified protein and less in Q78 K2932D/K2934D-purified proteins. The experiment was repeated three times.

    Article Snippet: Three lysine aspartic acid (KD) mutant huntingtin constructs (K2449D, K2932D/K2934D, K2449D/K2932D/K2934D) were generated by submission of the DNA sequences to Genscript (Piscataway, NJ, USA), which provided synthesized DNA in the pFastBac1 vector using SalI/SacII restriction digest and standard molecular biology techniques.

    Techniques: Mutagenesis, Binding Assay, Methylation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Western Blot

    The bbb22 gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. DNA was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.

    Journal: Infection and Immunity

    Article Title: Molecular Dissection of a Borrelia burgdorferiIn Vivo Essential Purine Transport System

    doi: 10.1128/IAI.02859-14

    Figure Lengend Snippet: The bbb22 gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. DNA was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.

    Article Snippet: Plasmids pBSV2G 22dist p - bbb22 + and pBSV2G 22prox p - bbb22 + were confirmed by restriction digestion and DNA sequence analysis (Genewiz).

    Techniques: Infection, Isolation, Mouse Assay

    Telomere DNA G-quadruplex unfolding by arginine to alanine mutants of UP1+RGG monitored using CD spectroscopy. ( A, B ) Unfolding of K + form of Tel22 G-quadruplex DNA by TriRGG (A) and AllRGG (B) mutants. The G-quadruplex DNA was titrated with increasing molar excess of proteins. The black arrows in the spectra indicate the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( C ) The normalized ellipticity at 295 nm of Tel22 at the final titration step (at 1:6 molar ratio of DNA to protein) for UP1, AllRGG, TriRGG and UP1+RGG showing the relative foldedness of the G-quadruplex structure.

    Journal: Nucleic Acids Research

    Article Title: RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

    doi: 10.1093/nar/gky854

    Figure Lengend Snippet: Telomere DNA G-quadruplex unfolding by arginine to alanine mutants of UP1+RGG monitored using CD spectroscopy. ( A, B ) Unfolding of K + form of Tel22 G-quadruplex DNA by TriRGG (A) and AllRGG (B) mutants. The G-quadruplex DNA was titrated with increasing molar excess of proteins. The black arrows in the spectra indicate the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( C ) The normalized ellipticity at 295 nm of Tel22 at the final titration step (at 1:6 molar ratio of DNA to protein) for UP1, AllRGG, TriRGG and UP1+RGG showing the relative foldedness of the G-quadruplex structure.

    Article Snippet: DNA preparation for CD, NMR and fluorescence kinetics experiments The DNA sequences (Table ) for binding studies with UP1, UP1+RGG and the RGG-box were ordered from Eurofins except the abasicloops-Tel22 DNA that was ordered from Dharmacon.

    Techniques: Spectroscopy, Protein Concentration, Titration

    Interaction of RGG-box with the single stranded and G-quadruplex DNA monitored through NMR spectroscopy. ( A ) 2D 15 N– 1 H HSQC spectrum of the free RGG-box (black) and in complex with Tel22ss at 1:6 protein to DNA molar ratio (red). No significant chemical shift perturbations were observed for this interaction. Single stranded Tel22ss is shown as a cartoon. ( B ) 2D 15 N– 1 H HSQC spectrum of the RGG-box (black) and in complex with Tel22 at 1:6 protein to DNA molar ratio (red). Specific chemical shift perturbations were observed for several residues (marked with green arrows). A representative cartoon of monomeric G-quadruplex form of Tel22 is shown (only one conformation in K + ion is shown). ( C ) A subset of residues of RGG-box that show specific chemical shift perturbation upon addition of Tel22 is shown. The RGG-box and Tel22 complex was in fast exchange (weak binding) as we observed continuous movement of resonance peaks upon addition of increasing amount of the Tel22 DNA. Three steps of titration at different protein to DNA ratios are shown: black at 1:0, green at 1: 1, and red at 1:6. ( D ) The titration curves showing chemical shift change plotted as a function of increasing DNA:protein ratio for 14 interacting residues of the RGG-box is shown.

    Journal: Nucleic Acids Research

    Article Title: RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

    doi: 10.1093/nar/gky854

    Figure Lengend Snippet: Interaction of RGG-box with the single stranded and G-quadruplex DNA monitored through NMR spectroscopy. ( A ) 2D 15 N– 1 H HSQC spectrum of the free RGG-box (black) and in complex with Tel22ss at 1:6 protein to DNA molar ratio (red). No significant chemical shift perturbations were observed for this interaction. Single stranded Tel22ss is shown as a cartoon. ( B ) 2D 15 N– 1 H HSQC spectrum of the RGG-box (black) and in complex with Tel22 at 1:6 protein to DNA molar ratio (red). Specific chemical shift perturbations were observed for several residues (marked with green arrows). A representative cartoon of monomeric G-quadruplex form of Tel22 is shown (only one conformation in K + ion is shown). ( C ) A subset of residues of RGG-box that show specific chemical shift perturbation upon addition of Tel22 is shown. The RGG-box and Tel22 complex was in fast exchange (weak binding) as we observed continuous movement of resonance peaks upon addition of increasing amount of the Tel22 DNA. Three steps of titration at different protein to DNA ratios are shown: black at 1:0, green at 1: 1, and red at 1:6. ( D ) The titration curves showing chemical shift change plotted as a function of increasing DNA:protein ratio for 14 interacting residues of the RGG-box is shown.

    Article Snippet: DNA preparation for CD, NMR and fluorescence kinetics experiments The DNA sequences (Table ) for binding studies with UP1, UP1+RGG and the RGG-box were ordered from Eurofins except the abasicloops-Tel22 DNA that was ordered from Dharmacon.

    Techniques: Nuclear Magnetic Resonance, Spectroscopy, Binding Assay, Titration

    Telomere DNA G-quadruplex unfolding by UP1+RGG and UP1 monitored using CD spectroscopy. ( A, B ) Unfolding of K + and Na + forms of Tel22 G-quadruplex DNA by UP1. The G-quadruplex DNAs were titrated with increasing molar excess of proteins. The black arrow in the spectra indicates the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( C, D ) Unfolding of K + and Na + forms of Tel22 G-quadruplex DNA by UP1+RGG. The G-quadruplex DNAs were titrated with increasing molar excess of proteins. The black arrow in the spectra indicates the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( E, F ) The ellipticity at 295 nm was normalized and plotted to show the relative foldedness of both K + and Na + forms of quadruplexes upon UP1 or UP1+RGG addition at each step of titration.

    Journal: Nucleic Acids Research

    Article Title: RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

    doi: 10.1093/nar/gky854

    Figure Lengend Snippet: Telomere DNA G-quadruplex unfolding by UP1+RGG and UP1 monitored using CD spectroscopy. ( A, B ) Unfolding of K + and Na + forms of Tel22 G-quadruplex DNA by UP1. The G-quadruplex DNAs were titrated with increasing molar excess of proteins. The black arrow in the spectra indicates the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( C, D ) Unfolding of K + and Na + forms of Tel22 G-quadruplex DNA by UP1+RGG. The G-quadruplex DNAs were titrated with increasing molar excess of proteins. The black arrow in the spectra indicates the gradual decrease in ellipticity at 295 nm with increasing protein concentration. ( E, F ) The ellipticity at 295 nm was normalized and plotted to show the relative foldedness of both K + and Na + forms of quadruplexes upon UP1 or UP1+RGG addition at each step of titration.

    Article Snippet: DNA preparation for CD, NMR and fluorescence kinetics experiments The DNA sequences (Table ) for binding studies with UP1, UP1+RGG and the RGG-box were ordered from Eurofins except the abasicloops-Tel22 DNA that was ordered from Dharmacon.

    Techniques: Spectroscopy, Protein Concentration, Titration

    Telomere DNA G-quadruplex unfolding by UP1+RGG and UP1 monitored using NMR and fluorescence spectroscopy. ( A ) 1D 1 H NMR spectra of Na + form of Tel22 showing gradual loss of imino proton peaks upon titration with increasing concentrations of UP1 (blue) and UP1+RGG (red). ( B ) Unfolding of the 5′-FAM and 3′-TAMRA labeled K + form of Tel22 DNA G-quadruplex (5′FAM-Tel22-TAMRA3′) by UP1 (blue) and UP1+RGG (red) monitored by observing the emission of FAM at 516 nM. 5′FAM-Tel22-TAMRA3′ DNA was mixed with 4 molar equivalents of UP1 or UP1+RGG and the emission spectrum was recorded over a time period. ( C ) Proposed model for RGG-box assisted recognition and unfolding of telomere DNA G-quadruplex unfolding by UP1+RGG.

    Journal: Nucleic Acids Research

    Article Title: RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

    doi: 10.1093/nar/gky854

    Figure Lengend Snippet: Telomere DNA G-quadruplex unfolding by UP1+RGG and UP1 monitored using NMR and fluorescence spectroscopy. ( A ) 1D 1 H NMR spectra of Na + form of Tel22 showing gradual loss of imino proton peaks upon titration with increasing concentrations of UP1 (blue) and UP1+RGG (red). ( B ) Unfolding of the 5′-FAM and 3′-TAMRA labeled K + form of Tel22 DNA G-quadruplex (5′FAM-Tel22-TAMRA3′) by UP1 (blue) and UP1+RGG (red) monitored by observing the emission of FAM at 516 nM. 5′FAM-Tel22-TAMRA3′ DNA was mixed with 4 molar equivalents of UP1 or UP1+RGG and the emission spectrum was recorded over a time period. ( C ) Proposed model for RGG-box assisted recognition and unfolding of telomere DNA G-quadruplex unfolding by UP1+RGG.

    Article Snippet: DNA preparation for CD, NMR and fluorescence kinetics experiments The DNA sequences (Table ) for binding studies with UP1, UP1+RGG and the RGG-box were ordered from Eurofins except the abasicloops-Tel22 DNA that was ordered from Dharmacon.

    Techniques: Nuclear Magnetic Resonance, Fluorescence, Spectroscopy, Titration, Labeling

    Interaction of UP1+RGG and UP1 with the single stranded and G-quadruplex DNA monitored through ITC. Raw and fitted isotherms are shown and the equilibrium K d s obtained upon fitting of the raw data is mentioned in each panel. ( A, B ) Interaction of UP1 with the single stranded Tel22ss DNA in the presence of 100 mM NaCl and 100 mM KCl respectively. ( C, D ) Interaction of UP1+RGG with the single stranded Tel22ss DNA in the presence of 100 mM NaCl and 100 mM KCl respectively. ( E, F ) Interaction of UP1 with the Na + and K + forms of Tel22 G-quadruplex DNA respectively. ( G, H ) Interaction of UP1+RGG with the Na + and K + forms of Tel22 G-quadruplex DNA respectively.

    Journal: Nucleic Acids Research

    Article Title: RGG-box in hnRNPA1 specifically recognizes the telomere G-quadruplex DNA and enhances the G-quadruplex unfolding ability of UP1 domain

    doi: 10.1093/nar/gky854

    Figure Lengend Snippet: Interaction of UP1+RGG and UP1 with the single stranded and G-quadruplex DNA monitored through ITC. Raw and fitted isotherms are shown and the equilibrium K d s obtained upon fitting of the raw data is mentioned in each panel. ( A, B ) Interaction of UP1 with the single stranded Tel22ss DNA in the presence of 100 mM NaCl and 100 mM KCl respectively. ( C, D ) Interaction of UP1+RGG with the single stranded Tel22ss DNA in the presence of 100 mM NaCl and 100 mM KCl respectively. ( E, F ) Interaction of UP1 with the Na + and K + forms of Tel22 G-quadruplex DNA respectively. ( G, H ) Interaction of UP1+RGG with the Na + and K + forms of Tel22 G-quadruplex DNA respectively.

    Article Snippet: DNA preparation for CD, NMR and fluorescence kinetics experiments The DNA sequences (Table ) for binding studies with UP1, UP1+RGG and the RGG-box were ordered from Eurofins except the abasicloops-Tel22 DNA that was ordered from Dharmacon.

    Techniques:

    Relationship between the Illumina DNA-sequencing read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

    doi: 10.1093/dnares/dsy005

    Figure Lengend Snippet: Relationship between the Illumina DNA-sequencing read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.

    Article Snippet: Recently, there are many plant genomes assembled by Illumina DNA-sequencing reads.

    Techniques: DNA Sequencing

    Three sets of OGGs among non- Arabidopsis species, A. thaliana , A. lyrata and A. halleri. OGGs between A. lyrata and A. halleri were defined as AL–AH OGGs. There were 25,833, 26,428 and 26,007 AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among A. thaliana , A. lyrata and A. halleri were defined as AT–AL–AH OGGs. There were 22,105, 22,684 and 21,537 AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among non- Arabidopsis species ( B. rapa , B. stricta , C. grandiflora , C. rubella , E. salsugineum ), A. thaliana , A. lyrata and A. halleri were defined as nonA-AT–AL–AH OGGs. There were 17,669 and 17,925 non-A-AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes and the available A. halleri genome, respectively.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

    doi: 10.1093/dnares/dsy005

    Figure Lengend Snippet: Three sets of OGGs among non- Arabidopsis species, A. thaliana , A. lyrata and A. halleri. OGGs between A. lyrata and A. halleri were defined as AL–AH OGGs. There were 25,833, 26,428 and 26,007 AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among A. thaliana , A. lyrata and A. halleri were defined as AT–AL–AH OGGs. There were 22,105, 22,684 and 21,537 AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among non- Arabidopsis species ( B. rapa , B. stricta , C. grandiflora , C. rubella , E. salsugineum ), A. thaliana , A. lyrata and A. halleri were defined as nonA-AT–AL–AH OGGs. There were 17,669 and 17,925 non-A-AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes and the available A. halleri genome, respectively.

    Article Snippet: Recently, there are many plant genomes assembled by Illumina DNA-sequencing reads.

    Techniques: DNA Sequencing

    Models for the multi-dimensional functions of TETs in mediating DNA methylation and hydroxymethylation. (A) TET family members enrich 5hmC at CGI shores to provide a protective boundary against aberrant hypermethylation (i). In the absence of TETs, 5hmC cannot be established, permitting the aberrant expansion of 5mC into the CGI (ii). (B) In the normal pluripotent state, TETs eliminate 5hmC from promoters and remove both 5mC and 5hmC from gene bodies of a subset of highly transcriptionally active H3K4me3- and H3K36me3-marked genes (i). TETs mediate hydroxymethylation and promote a low level of methylation at H3K27me3-marked promoters (ii). At H2AK119ub-marked promoters TET proteins enrich 5hmC and promote turnover of cytosine modifications by mediating demethylation of 5mC (iii). In the event of TET mutation or other TET functional disruption (like decreased expression), these distinct epigenetic patterns are lost, making loci vulnerable to changes in transcriptional activity, perhaps by leaving unmodified cytosines available for aberrant DNA methylation by DNMTs or by permitting the binding of chromatin remodelers or repressors such as PRC1 or PRC2.

    Journal: Genome Biology

    Article Title: Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells

    doi: 10.1186/gb-2014-15-6-r81

    Figure Lengend Snippet: Models for the multi-dimensional functions of TETs in mediating DNA methylation and hydroxymethylation. (A) TET family members enrich 5hmC at CGI shores to provide a protective boundary against aberrant hypermethylation (i). In the absence of TETs, 5hmC cannot be established, permitting the aberrant expansion of 5mC into the CGI (ii). (B) In the normal pluripotent state, TETs eliminate 5hmC from promoters and remove both 5mC and 5hmC from gene bodies of a subset of highly transcriptionally active H3K4me3- and H3K36me3-marked genes (i). TETs mediate hydroxymethylation and promote a low level of methylation at H3K27me3-marked promoters (ii). At H2AK119ub-marked promoters TET proteins enrich 5hmC and promote turnover of cytosine modifications by mediating demethylation of 5mC (iii). In the event of TET mutation or other TET functional disruption (like decreased expression), these distinct epigenetic patterns are lost, making loci vulnerable to changes in transcriptional activity, perhaps by leaving unmodified cytosines available for aberrant DNA methylation by DNMTs or by permitting the binding of chromatin remodelers or repressors such as PRC1 or PRC2.

    Article Snippet: DNA sequencing libraries were generated from the 5mC and 5hmC captured DNA with the TruSeq DNA sample preparation kit (Illumina, San Diego, CA USA) according to manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Methylation, Mutagenesis, Functional Assay, Expressing, Activity Assay, Binding Assay

    Depletion of TET1, TET2, or TET3 causes genome-wide loss of 5hmC and both DNA hypomethylation and hypermethylation. (A) Tag density plots of 5mC (dashed line plots) and 5hmC (solid line plots) from -5 to +5 kb across gene promoters, across gene bodies (25 to 75%), and from -5 to +5 kb across the transcription termination site (TTS) (left panels). Tag density plots were also drawn for exons across HCP, intermediate CpG density promoter (ICP), and LCP genes (right panels). (B) Pie charts for genes with decreased (top) and increased (bottom) 5hmC. Pie pieces represent total number of genes with two-fold or greater 5hmC change in the specified gene region. (C) Area proportional Venn diagrams illustrating overlap of promoters that lose 5hmC and gain or lose 5mC in each TET knockdown. P

    Journal: Genome Biology

    Article Title: Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells

    doi: 10.1186/gb-2014-15-6-r81

    Figure Lengend Snippet: Depletion of TET1, TET2, or TET3 causes genome-wide loss of 5hmC and both DNA hypomethylation and hypermethylation. (A) Tag density plots of 5mC (dashed line plots) and 5hmC (solid line plots) from -5 to +5 kb across gene promoters, across gene bodies (25 to 75%), and from -5 to +5 kb across the transcription termination site (TTS) (left panels). Tag density plots were also drawn for exons across HCP, intermediate CpG density promoter (ICP), and LCP genes (right panels). (B) Pie charts for genes with decreased (top) and increased (bottom) 5hmC. Pie pieces represent total number of genes with two-fold or greater 5hmC change in the specified gene region. (C) Area proportional Venn diagrams illustrating overlap of promoters that lose 5hmC and gain or lose 5mC in each TET knockdown. P

    Article Snippet: DNA sequencing libraries were generated from the 5mC and 5hmC captured DNA with the TruSeq DNA sample preparation kit (Illumina, San Diego, CA USA) according to manufacturer’s instructions.

    Techniques: Genome Wide

    Relationships between gene expression and DNA epigenetic marks. (A) Quantity of upregulated and downregulated genes in siTET1, siTET2, and siTET3 conditions. (B) NCCIT cell gene expression changes that occur with siTET depletions were compared to gene expression changes that occur during RA-induced differentiation from UD to DF. Downregulated genes in siTET1, siTET2, and siTET3 depleted cells were significantly enriched for genes upregulated during differentiation. Shown are the percentages of genes downregulated in siTET conditions that overlap with genes that become upregulated in DF cells and the percentage of total upregulated genes in DF cells. (C) Hypo- or hyper-5hmC introns of siTET depletions were compared to basal gene expression levels in UD NCCIT cells. Shown is the percentage of genes with 5hmC changes in introns in siTET-treated cells that occur in highly, moderately, or lowly expressed genes. These percentages of overlapping genes are compared to the total percentage of highly (red bar), moderately (gold bar), or lowly (green bar) expressed genes. (D) Area proportional Venn diagrams showing the number of repressed genes that are in proximity to H3K27ac-marked enhancers that lose 5hmC upon TET depletion (* P

    Journal: Genome Biology

    Article Title: Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells

    doi: 10.1186/gb-2014-15-6-r81

    Figure Lengend Snippet: Relationships between gene expression and DNA epigenetic marks. (A) Quantity of upregulated and downregulated genes in siTET1, siTET2, and siTET3 conditions. (B) NCCIT cell gene expression changes that occur with siTET depletions were compared to gene expression changes that occur during RA-induced differentiation from UD to DF. Downregulated genes in siTET1, siTET2, and siTET3 depleted cells were significantly enriched for genes upregulated during differentiation. Shown are the percentages of genes downregulated in siTET conditions that overlap with genes that become upregulated in DF cells and the percentage of total upregulated genes in DF cells. (C) Hypo- or hyper-5hmC introns of siTET depletions were compared to basal gene expression levels in UD NCCIT cells. Shown is the percentage of genes with 5hmC changes in introns in siTET-treated cells that occur in highly, moderately, or lowly expressed genes. These percentages of overlapping genes are compared to the total percentage of highly (red bar), moderately (gold bar), or lowly (green bar) expressed genes. (D) Area proportional Venn diagrams showing the number of repressed genes that are in proximity to H3K27ac-marked enhancers that lose 5hmC upon TET depletion (* P

    Article Snippet: DNA sequencing libraries were generated from the 5mC and 5hmC captured DNA with the TruSeq DNA sample preparation kit (Illumina, San Diego, CA USA) according to manufacturer’s instructions.

    Techniques: Expressing