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  • 99
    Millipore uridine
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    Millipore 5fdur
    In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with <t>5FdUR</t> show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).
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    Millipore anti flag antibody
    In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with <t>5FdUR</t> show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).
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    Millipore anti flag m2 antibody
    In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with <t>5FdUR</t> show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).
    Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lb luria bertani broth
    In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with <t>5FdUR</t> show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).
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    Image Search Results


    In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with 5FdUR show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: In situ detection of the cellular endogenous U-DNA content. (A) Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with 5FdUR show efficient staining with the uracil sensor compared to non-treated cells. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with PFA fixation compared to the Carnoy fixation applied previously ( Róna et al., 2016 ). Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest ( Huehls et al., 2016 ; Yan et al., 2016 ).

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: In Situ, Construct, Immunocytochemistry, FLAG-tag, Expressing, Staining

    Comparison of processed U-DNA-Seq data among samples (A) Representative IGV view on the log2 ratio and the derived regions of uracil enrichment (two replicates for each sample were merged). Log2 ratio signal tracks of enriched versus input coverage (log2, upper track) and derived regions of uracil enrichment (regions, bottom track) for non-treated: wild type (WT, red) and UGI-expressing (NT_UGI, orange); and for treated: with 5FdUR (5FdUR_UGI, green) or raltitrexed (RTX_UGI, blue) HCT116 samples are shown in genomic segment (chr2:64,500,000-89,500,001). Differences between treated and non-treated samples are clearly visible. Furthermore, 5FdUR and RTX treatments caused similar but not identical uracil enrichment profile (differences are highlighted with yellow background). (B) Comparison of log2 uracil enrichment profiles among samples was performed using multiBigWigSummary (deepTools) and Pearson correlation were plotted using plotCorrelation (deepTools). A heatmap combined with scatterplots is shown for the four samples. (C) Histograms of log2 ratio profiles were calculated and plotted using R. A sub-population of data bins with elevated log2 uracil enrichment signal is clearly visible (indicated with asterisk) in case of drug-treated samples, where high uracil incorporation was detected (cf. Supplementary Figure S1B-C). Thresholds applied in determination of uracil enriched regions are indicated with red line (cf. Supplementary Table S3A).

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: Comparison of processed U-DNA-Seq data among samples (A) Representative IGV view on the log2 ratio and the derived regions of uracil enrichment (two replicates for each sample were merged). Log2 ratio signal tracks of enriched versus input coverage (log2, upper track) and derived regions of uracil enrichment (regions, bottom track) for non-treated: wild type (WT, red) and UGI-expressing (NT_UGI, orange); and for treated: with 5FdUR (5FdUR_UGI, green) or raltitrexed (RTX_UGI, blue) HCT116 samples are shown in genomic segment (chr2:64,500,000-89,500,001). Differences between treated and non-treated samples are clearly visible. Furthermore, 5FdUR and RTX treatments caused similar but not identical uracil enrichment profile (differences are highlighted with yellow background). (B) Comparison of log2 uracil enrichment profiles among samples was performed using multiBigWigSummary (deepTools) and Pearson correlation were plotted using plotCorrelation (deepTools). A heatmap combined with scatterplots is shown for the four samples. (C) Histograms of log2 ratio profiles were calculated and plotted using R. A sub-population of data bins with elevated log2 uracil enrichment signal is clearly visible (indicated with asterisk) in case of drug-treated samples, where high uracil incorporation was detected (cf. Supplementary Figure S1B-C). Thresholds applied in determination of uracil enriched regions are indicated with red line (cf. Supplementary Table S3A).

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: DNA Sequencing, Derivative Assay, Expressing

    Characterization of U-DNA enrichment patterns. (A) Top hs from GIGGLE search on HCT116 specific dataset. GIGGLE search was performed with interval (bed) files of uracil enriched regions on a set of HCT116 related ChIP-seq and DIP-seq experiment data (for details see the Supplementary Material). Factors corresponding to the top 10 hits for each sample were selected. GIGGLE scores between all four samples and all experiments corresponding to these factors were plotted excluding CNOT3, H2B, H3K27me1/2 where data were not informative (data are found in Supplementary Material Appendix 2). Histone marks and the only transcription factor, SP1 are categorized depending on their occurrence in transcriptionally active or repressive regions. Notably, some of them have plastic behaviour allowing either transcriptionally active or repressive function. U-DNA-Seq samples are as follows: non-treated wild type (WT, red), non-treated UGI-expressing (NT_UGI, orange), 5FdUR treated UGI-expressing (5FdUR_UGI, green), and RTX treated UGI-expressing (RTX_UGI, blue) HCT116 cells. (B) Correlation with genomic features. Interval (bed) files of genomic features were obtained from UCSC, Ensembl, and ReplicationDomain databases (for details see the Supplementary Material), and correlation with interval files of uracil regions were analysed using bedtools annotate software. Numbers of overlapping basepairs were summarized for each pair of interval files, and scores were calculated according the formula: (baseNo_overlap/baseNo_sample_file) * (baseNo_overlap/baseNo_feature_file) * 10000. Heatmaps were created based on fold increase of the scores compared to the corresponding WT scores. Sizes of interval files in number of basepairs are also given in the second column and the second line. Upon drug treatments, a clear shift from non-coding / heterochromatic / late replicated segments towards more active / coding / euchromatic / early replicated segments can be seen. CDS, coding sequence; SINE, short interspersed element; LTR, long terminal repeat; LINE, long interspersed element; cytoBand, cytogenic chromosome band negatively (gneg) or positively (gpos) stained by Giemsa; RT, replication timing; DNaseHS, DNase hypersensitive site. (C) Correlation analysis with replication timing. Replication timing data (bigWig files with 5000 bp binsize) specific for HCT116 were downloaded from ReplicationDomain database. Data bins were distributed to 10 equal size groups according to replication timing from early to late. Then log2 uracil enrichment signals for these data bin groups were plotted for each sample using R.

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: Characterization of U-DNA enrichment patterns. (A) Top hs from GIGGLE search on HCT116 specific dataset. GIGGLE search was performed with interval (bed) files of uracil enriched regions on a set of HCT116 related ChIP-seq and DIP-seq experiment data (for details see the Supplementary Material). Factors corresponding to the top 10 hits for each sample were selected. GIGGLE scores between all four samples and all experiments corresponding to these factors were plotted excluding CNOT3, H2B, H3K27me1/2 where data were not informative (data are found in Supplementary Material Appendix 2). Histone marks and the only transcription factor, SP1 are categorized depending on their occurrence in transcriptionally active or repressive regions. Notably, some of them have plastic behaviour allowing either transcriptionally active or repressive function. U-DNA-Seq samples are as follows: non-treated wild type (WT, red), non-treated UGI-expressing (NT_UGI, orange), 5FdUR treated UGI-expressing (5FdUR_UGI, green), and RTX treated UGI-expressing (RTX_UGI, blue) HCT116 cells. (B) Correlation with genomic features. Interval (bed) files of genomic features were obtained from UCSC, Ensembl, and ReplicationDomain databases (for details see the Supplementary Material), and correlation with interval files of uracil regions were analysed using bedtools annotate software. Numbers of overlapping basepairs were summarized for each pair of interval files, and scores were calculated according the formula: (baseNo_overlap/baseNo_sample_file) * (baseNo_overlap/baseNo_feature_file) * 10000. Heatmaps were created based on fold increase of the scores compared to the corresponding WT scores. Sizes of interval files in number of basepairs are also given in the second column and the second line. Upon drug treatments, a clear shift from non-coding / heterochromatic / late replicated segments towards more active / coding / euchromatic / early replicated segments can be seen. CDS, coding sequence; SINE, short interspersed element; LTR, long terminal repeat; LINE, long interspersed element; cytoBand, cytogenic chromosome band negatively (gneg) or positively (gpos) stained by Giemsa; RT, replication timing; DNaseHS, DNase hypersensitive site. (C) Correlation analysis with replication timing. Replication timing data (bigWig files with 5000 bp binsize) specific for HCT116 were downloaded from ReplicationDomain database. Data bins were distributed to 10 equal size groups according to replication timing from early to late. Then log2 uracil enrichment signals for these data bin groups were plotted for each sample using R.

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: Chromatin Immunoprecipitation, DNA Immunoprecipitation Sequencing, DNA Sequencing, Expressing, Software, Sequencing, Staining

    Genomic uracil moieties colocalize with H3K36me3 and H3K27me3 analysed by super-resolution microscopy. Confocal and dSTORM imaging were performed on non-treated, 5FdUR or RTX treated HCT116 cells stably expressing UGI to compare the localization of genomic uracil residues (red) to histone markers, H3K36me3 (green) (A) or H3K27me3 (green) (B) , selected based on the U-DNA-Seq results. Scale bar represents 5 μm. The graphs display the Cross-Pair-Correlation analysis between U-DNA and H3K36me3 (C) or H3K27me3 (D) , respectively. Overlap is defined as any amount of pixel overlap between segmented objects. Total number of analysed nuclei for H3K36me3 staining (C) were the following: NT_UGI (n=205), 5FdUR_UGI (n=101) and RTX_UGI (n=153) from 2 independent experiments. Total number of analysed nuclei for H3K27me3 staining (D) were the following: NT_UGI (n=154), 5FdUR_UGI (n=151) and RTX_UGI (n=107) from 2 independent experiments. Black line denotes the mean of each dataset. The colour code follows the one in Figure 3A .

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: Genomic uracil moieties colocalize with H3K36me3 and H3K27me3 analysed by super-resolution microscopy. Confocal and dSTORM imaging were performed on non-treated, 5FdUR or RTX treated HCT116 cells stably expressing UGI to compare the localization of genomic uracil residues (red) to histone markers, H3K36me3 (green) (A) or H3K27me3 (green) (B) , selected based on the U-DNA-Seq results. Scale bar represents 5 μm. The graphs display the Cross-Pair-Correlation analysis between U-DNA and H3K36me3 (C) or H3K27me3 (D) , respectively. Overlap is defined as any amount of pixel overlap between segmented objects. Total number of analysed nuclei for H3K36me3 staining (C) were the following: NT_UGI (n=205), 5FdUR_UGI (n=101) and RTX_UGI (n=153) from 2 independent experiments. Total number of analysed nuclei for H3K27me3 staining (D) were the following: NT_UGI (n=154), 5FdUR_UGI (n=151) and RTX_UGI (n=107) from 2 independent experiments. Black line denotes the mean of each dataset. The colour code follows the one in Figure 3A .

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: Microscopy, Imaging, Stable Transfection, Expressing, DNA Sequencing, Staining

    The FLAG-ΔUNG-SNAP sensor enables super-resolution detection of genomic uracil by STED and dSTORM microscopy. (A) U-DNA staining was performed on non-treated or 5FdUR treated HCT116 cells stably expressing UGI. Different SNAP-tag substrates, SNAP647 for confocal and SNAP546 for super-resolution imaging (STED) were used to label FLAG-ΔUNG-SNAP. Scale bar represents 20 μm for whole images and 10 μm for zoomed sections. (B) dSTORM imaging was also performed on non-treated or drug-treated (5FdUR or RTX) HCT116 cells stably expressing UGI to compare the sensitivity of these imaging techniques. U-DNA staining shows a characteristic distribution pattern in cells with elevated uracil levels as compared to non-treated cells. SNAP647 substrate was used to label FLAG-ΔUNG-SNAP. Scale bar represents 10 μm for whole images and 2 μm for zoomed sections.

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: The FLAG-ΔUNG-SNAP sensor enables super-resolution detection of genomic uracil by STED and dSTORM microscopy. (A) U-DNA staining was performed on non-treated or 5FdUR treated HCT116 cells stably expressing UGI. Different SNAP-tag substrates, SNAP647 for confocal and SNAP546 for super-resolution imaging (STED) were used to label FLAG-ΔUNG-SNAP. Scale bar represents 20 μm for whole images and 10 μm for zoomed sections. (B) dSTORM imaging was also performed on non-treated or drug-treated (5FdUR or RTX) HCT116 cells stably expressing UGI to compare the sensitivity of these imaging techniques. U-DNA staining shows a characteristic distribution pattern in cells with elevated uracil levels as compared to non-treated cells. SNAP647 substrate was used to label FLAG-ΔUNG-SNAP. Scale bar represents 10 μm for whole images and 2 μm for zoomed sections.

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: Microscopy, Staining, Stable Transfection, Expressing, Imaging

    U-DNA-Seq provides genome-wide mapping of uracil-DNA distribution. (A) Schematic image of the novel U-DNA immunoprecipitation and sequencing method (U-DNA-Seq). After sonication, enrichment of the fragmented U-DNA was carried out by the 1xFLAG-ΔUNG sensor construct followed by pull-down with anti-FLAG agarose beads. U-DNA enrichment compared to input DNA was confirmed by dot blot assay before samples were subjected to NGS. (B) Immunoprecipitation led to elevated uracil levels in enriched U-DNA samples compared to input DNA in case of both 5FdUR (5FdUR_UGI) and RTX (RTX_UGI) treated samples. In case of the given treatment, the same amount of DNA was loaded from input and enriched U-DNA samples providing correct visual comparison of the dots. Two-third serial dilutions were applied.

    Journal: bioRxiv

    Article Title: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

    doi: 10.1101/2020.03.04.976977

    Figure Lengend Snippet: U-DNA-Seq provides genome-wide mapping of uracil-DNA distribution. (A) Schematic image of the novel U-DNA immunoprecipitation and sequencing method (U-DNA-Seq). After sonication, enrichment of the fragmented U-DNA was carried out by the 1xFLAG-ΔUNG sensor construct followed by pull-down with anti-FLAG agarose beads. U-DNA enrichment compared to input DNA was confirmed by dot blot assay before samples were subjected to NGS. (B) Immunoprecipitation led to elevated uracil levels in enriched U-DNA samples compared to input DNA in case of both 5FdUR (5FdUR_UGI) and RTX (RTX_UGI) treated samples. In case of the given treatment, the same amount of DNA was loaded from input and enriched U-DNA samples providing correct visual comparison of the dots. Two-third serial dilutions were applied.

    Article Snippet: Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 h either in the absence or presence of 20 μM 5FdUR (Sigma) before collecting them for genomic DNA purification.

    Techniques: DNA Sequencing, Genome Wide, Immunoprecipitation, Sequencing, Sonication, Construct, Dot Blot, Next-Generation Sequencing