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    New England Biolabs nlaiii
    Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) <t>SbfI-SphI</t> and ( B ) <t>PstI-NlaIII.</t>
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    Images

    1) Product Images from "Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)"

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    Journal: Animals : an Open Access Journal from MDPI

    doi: 10.3390/ani11030899

    Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) SbfI-SphI and ( B ) PstI-NlaIII.
    Figure Legend Snippet: Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Techniques Used:

    Manhattan and quantile–quantile plots of the association tests for length and log 2 K in the PstI-NlaIII scenario ( n = 179). The red horizontal line indicates the Bonferroni error rate-adjusted significance level. The blue line indicates the threshold of the significant markers after BH adjustment of p -values.
    Figure Legend Snippet: Manhattan and quantile–quantile plots of the association tests for length and log 2 K in the PstI-NlaIII scenario ( n = 179). The red horizontal line indicates the Bonferroni error rate-adjusted significance level. The blue line indicates the threshold of the significant markers after BH adjustment of p -values.

    Techniques Used:

    Distributions of post-filtering minor allele frequency (MAF) and single nucleotide polymorphism (SNP) call rate for SbfI-SphI ( n = 253) and the intersecting ( n = 175) animals that were also genotyped with PstI-NlaIII.
    Figure Legend Snippet: Distributions of post-filtering minor allele frequency (MAF) and single nucleotide polymorphism (SNP) call rate for SbfI-SphI ( n = 253) and the intersecting ( n = 175) animals that were also genotyped with PstI-NlaIII.

    Techniques Used:

    Heatmaps visualizing the relative frequencies (%) of family predictions according to the DAPC cross-validation scheme for ( A ) SbfI-SphI and ( B ) PstI-NlaIII.
    Figure Legend Snippet: Heatmaps visualizing the relative frequencies (%) of family predictions according to the DAPC cross-validation scheme for ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Techniques Used:

    Discriminant analysis of principal components (DAPC) for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.
    Figure Legend Snippet: Discriminant analysis of principal components (DAPC) for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Techniques Used:

    Principal component analysis for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.
    Figure Legend Snippet: Principal component analysis for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Techniques Used:

    2) Product Images from "The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination"

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

    Journal: Nature

    doi: 10.1038/s41586-019-1547-y

    Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.
    Figure Legend Snippet: Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.

    Techniques Used: Activity Assay, Blocking Assay, Two Tailed Test

    3) Product Images from "The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination"

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

    Journal: Nature

    doi: 10.1038/s41586-019-1547-y

    Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.
    Figure Legend Snippet: Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.

    Techniques Used: Activity Assay, Blocking Assay, Two Tailed Test

    4) Product Images from "Short interspersed elements (SINEs) are a major source of canine genomic diversity"

    Article Title: Short interspersed elements (SINEs) are a major source of canine genomic diversity

    Journal: Genome Research

    doi: 10.1101/gr.3765505

    Construction of libraries that are enriched for SINEC_Cf elements and flanking sequence. ( A ) Genomic DNA is cleaved with the frequently cutting restriction enzyme, NlaIII. ( B ) The cleaved fragments are self-ligated. ( C ) The circularized products are subjected to PCR using SINEC_Cf-specific primers. ( D ) The linear products are size-selected and cloned in a plasmid vector. ( E ) Inserts are sequenced with a vector-specific primer.
    Figure Legend Snippet: Construction of libraries that are enriched for SINEC_Cf elements and flanking sequence. ( A ) Genomic DNA is cleaved with the frequently cutting restriction enzyme, NlaIII. ( B ) The cleaved fragments are self-ligated. ( C ) The circularized products are subjected to PCR using SINEC_Cf-specific primers. ( D ) The linear products are size-selected and cloned in a plasmid vector. ( E ) Inserts are sequenced with a vector-specific primer.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation

    5) Product Images from "Transposon insertional mutagenesis of diverse yeast strains suggests coordinated gene essentiality polymorphisms"

    Article Title: Transposon insertional mutagenesis of diverse yeast strains suggests coordinated gene essentiality polymorphisms

    Journal: Nature Communications

    doi: 10.1038/s41467-022-29228-1

    Probing yeast gene essentiality polymorphism using transposon insertional mutagenesis. a Outline of the experimental procedure. Endogenous ADE2 and URA3 genes are deleted (indicated by a cross) in all yeast strains. Plasmid pBK257 that carries the selectable marker URA3 , inducible transposase gene Ac under the control of the GAL1 promoter, and transposon MiniDs that interrupts ADE2 is transformed into yeast cells. Cells grown on SD + galactose − adenine plates have the transposon excised from the plasmid and potentially randomly inserted into the yeast genome. Genomic DNA is extracted and digested with four-cutter restriction enzymes DpnII and NlaIII in parallel, followed by ligase-mediated circularization. Circular DNA is amplified using primer P5_MiniDs and P7_MiniDs to enrich transposon/chromosomal junction regions. Primer custom_P1 is for sequencing the flanking regions of MiniDs , while primer custom_P2 is for reading the 8-nucleotide index in the P7_MiniDs primer. b Neighbor-joining tree of the 16 S. cerevisiae strains used in the study. The tree is based on 179,416 single nucleotide polymorphisms (SNPs) identified in these strains and the scale bar represents 1 SNP per kb genomic sequence. Geographic locations and ecological origins of the strains as well as clade names (in black letters) are indicated. Bootstrap percentages are shown at interior branches. c Three examples of chromosomal segments showing genes and transposon insertions in S288C. The numbers after the chromosome number indicate genomic locations in nucleotides. Each vertical gray line represents one transposon insertion and the darkness of the line is proportional to the number of sequencing reads. Horizontal bars mark gene locations, with gene names provided below the bars and white arrows indicating transcriptional directions. Gene deletion-based essentiality annotations are shown by the color of the gene: red for essential and blue for nonessential. d Frequency distributions of the number of transposons per annotated essential gene (red), nonessential gene (blue), and intergenic region (gray) in S288C. Vertical dashed lines indicate medians of the corresponding distributions. e Frequency distributions of transposon density in annotated essential genes (red), nonessential genes (blue), and intergenic regions (gray) in S288C. f The receiver operating characteristic (ROC) curve analysis of predictions of S288C gene essentiality by the RF classifier. FPR false-positive rate, TPR true-positive rate, AUC area under the curve. g Frequency distribution of the RF predictions of essentiality for genes in the testing data. h Annotated and RF predicted essentialities of 5,058 genes in S288C. E essential, NE nonessential. Source data are provided as a Source Data file.
    Figure Legend Snippet: Probing yeast gene essentiality polymorphism using transposon insertional mutagenesis. a Outline of the experimental procedure. Endogenous ADE2 and URA3 genes are deleted (indicated by a cross) in all yeast strains. Plasmid pBK257 that carries the selectable marker URA3 , inducible transposase gene Ac under the control of the GAL1 promoter, and transposon MiniDs that interrupts ADE2 is transformed into yeast cells. Cells grown on SD + galactose − adenine plates have the transposon excised from the plasmid and potentially randomly inserted into the yeast genome. Genomic DNA is extracted and digested with four-cutter restriction enzymes DpnII and NlaIII in parallel, followed by ligase-mediated circularization. Circular DNA is amplified using primer P5_MiniDs and P7_MiniDs to enrich transposon/chromosomal junction regions. Primer custom_P1 is for sequencing the flanking regions of MiniDs , while primer custom_P2 is for reading the 8-nucleotide index in the P7_MiniDs primer. b Neighbor-joining tree of the 16 S. cerevisiae strains used in the study. The tree is based on 179,416 single nucleotide polymorphisms (SNPs) identified in these strains and the scale bar represents 1 SNP per kb genomic sequence. Geographic locations and ecological origins of the strains as well as clade names (in black letters) are indicated. Bootstrap percentages are shown at interior branches. c Three examples of chromosomal segments showing genes and transposon insertions in S288C. The numbers after the chromosome number indicate genomic locations in nucleotides. Each vertical gray line represents one transposon insertion and the darkness of the line is proportional to the number of sequencing reads. Horizontal bars mark gene locations, with gene names provided below the bars and white arrows indicating transcriptional directions. Gene deletion-based essentiality annotations are shown by the color of the gene: red for essential and blue for nonessential. d Frequency distributions of the number of transposons per annotated essential gene (red), nonessential gene (blue), and intergenic region (gray) in S288C. Vertical dashed lines indicate medians of the corresponding distributions. e Frequency distributions of transposon density in annotated essential genes (red), nonessential genes (blue), and intergenic regions (gray) in S288C. f The receiver operating characteristic (ROC) curve analysis of predictions of S288C gene essentiality by the RF classifier. FPR false-positive rate, TPR true-positive rate, AUC area under the curve. g Frequency distribution of the RF predictions of essentiality for genes in the testing data. h Annotated and RF predicted essentialities of 5,058 genes in S288C. E essential, NE nonessential. Source data are provided as a Source Data file.

    Techniques Used: Mutagenesis, Plasmid Preparation, Marker, Transformation Assay, Amplification, Sequencing

    6) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    7) Product Images from "Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and co-infections of multiple respiratory viruses by Rapid field-deployable sequencing"

    Article Title: Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and co-infections of multiple respiratory viruses by Rapid field-deployable sequencing

    Journal: medRxiv

    doi: 10.1101/2020.06.12.20129247

    Agarose gel electrophoresis results of singleplex RPA a , Agarose gel electrophoresis results of singleplex RPA with selected primers shown next a molecular size marker. The amplicons range from 194 bp to 466 bp. b , Agarose gel electrophoresis results of restriction enzyme digestion. The amplicon of pair 5 was digested by SpeI while the others were digested by NlaIII. The digested DNA bands (asterisks) were of expected sizes. c , Agarose gel electrophoresis results showing the sensitivity of RPA in amplifying the SARS-CoV-2 genome. Primer pair 4 was used in the experiment. Reliable amplification can be achieved with 1.4 copies (calculated from dilution) of the SARS-CoV-2 genome. d , Agarose gel electrophoresis result of one-pot reverse transcription and RPA reaction using primer pair 4.
    Figure Legend Snippet: Agarose gel electrophoresis results of singleplex RPA a , Agarose gel electrophoresis results of singleplex RPA with selected primers shown next a molecular size marker. The amplicons range from 194 bp to 466 bp. b , Agarose gel electrophoresis results of restriction enzyme digestion. The amplicon of pair 5 was digested by SpeI while the others were digested by NlaIII. The digested DNA bands (asterisks) were of expected sizes. c , Agarose gel electrophoresis results showing the sensitivity of RPA in amplifying the SARS-CoV-2 genome. Primer pair 4 was used in the experiment. Reliable amplification can be achieved with 1.4 copies (calculated from dilution) of the SARS-CoV-2 genome. d , Agarose gel electrophoresis result of one-pot reverse transcription and RPA reaction using primer pair 4.

    Techniques Used: Agarose Gel Electrophoresis, Recombinase Polymerase Amplification, Marker, Amplification

    8) Product Images from "COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance"

    Article Title: COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24078-9

    COVseq implementation and validation. a Location along the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome (top) of MseI and NlaIII recognition sites (vertical black bars) and Centers for Disease Control and Prevention (CDC) multiplexed PCR assay amplicon pools (colored rectangles). Gene names (top) are according to the reference SARS-CoV-2 sequence NC_045512.2. b Schematic high-throughput COVseq workflow. Purified RNA samples (e.g., extracted from nasal- or oro-pharyngeal swabs) are first equally distributed in corresponding wells of six 96-well plates and amplified using six different PCR primer pools (one pool per plate) to amplify the amplicons shown in ( a ). After PCR, the contents of the wells in the six 96-well plates are pooled into the corresponding wells of a new 96-well plate and purified. Afterwards, 96 CUTseq adapters (see Supplementary Data 2 ) are used to barcode each sample individually, before all the samples are pooled together into the same sequencing library. Alternatively, 384 samples can be barcoded separately before being pooled together, by using the 384 CUTseq adapters listed in Supplementary Data 2 . c Percentage of bases in the SARS-CoV-2 reference genome covered by COVseq at varying sequencing depths (SE150 sequencing) for three different libraries prepared from RNA extracted from the supernatant of a viral culture, using genome digestion with one or two restriction enzymes (MseI and NlaIII). d Same as in ( c ), but for the S gene encoding the spike protein. e Inverse correlation between the cycle threshold (Ct) determined by RT-PCR and the number of reads, for OAS-29 samples (see Supplementary Data 4 ) sequenced by COVseq (MiSeq PE300). f Correlation between the total number of reads obtained with COVseq vs. NEBNext for the same samples in ( e ). g Correlation between the breadth of coverage at 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × sequencing depth obtained by COVseq vs. NEBNext for the same samples in ( e ). h Percentage of sequencing reads aligned to the SARS-CoV-2 reference genome, human reference genome (Hs), other genomes or unmapped, for the same samples in ( e ). The bottom plot shows the Ct value of each sample. i Correlation between the number of single-nucleotide variants (SNVs) per sample detected by COVseq (PE300) vs. NEBNext (SE75) in 20 ( n ) out of 29 OAS-29 samples with Ct ≤ 35. j Matrix showing the SNVs detected by COVseq, NEBNext, or both in the 20 OAS-29 samples with Ct ≤ 35. k Heatmap of the depth of coverage at the genomic positions of all the SNVs defining the UK (B.1.1.7), South African (B.1.351) and Brazilian (P.1) variants of concern (VOC) for the 20 OAS-29 samples with Ct ≤ 35 sequenced by COVseq. Gray color indicates locations that would have insufficient coverage to call SNVs (
    Figure Legend Snippet: COVseq implementation and validation. a Location along the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome (top) of MseI and NlaIII recognition sites (vertical black bars) and Centers for Disease Control and Prevention (CDC) multiplexed PCR assay amplicon pools (colored rectangles). Gene names (top) are according to the reference SARS-CoV-2 sequence NC_045512.2. b Schematic high-throughput COVseq workflow. Purified RNA samples (e.g., extracted from nasal- or oro-pharyngeal swabs) are first equally distributed in corresponding wells of six 96-well plates and amplified using six different PCR primer pools (one pool per plate) to amplify the amplicons shown in ( a ). After PCR, the contents of the wells in the six 96-well plates are pooled into the corresponding wells of a new 96-well plate and purified. Afterwards, 96 CUTseq adapters (see Supplementary Data 2 ) are used to barcode each sample individually, before all the samples are pooled together into the same sequencing library. Alternatively, 384 samples can be barcoded separately before being pooled together, by using the 384 CUTseq adapters listed in Supplementary Data 2 . c Percentage of bases in the SARS-CoV-2 reference genome covered by COVseq at varying sequencing depths (SE150 sequencing) for three different libraries prepared from RNA extracted from the supernatant of a viral culture, using genome digestion with one or two restriction enzymes (MseI and NlaIII). d Same as in ( c ), but for the S gene encoding the spike protein. e Inverse correlation between the cycle threshold (Ct) determined by RT-PCR and the number of reads, for OAS-29 samples (see Supplementary Data 4 ) sequenced by COVseq (MiSeq PE300). f Correlation between the total number of reads obtained with COVseq vs. NEBNext for the same samples in ( e ). g Correlation between the breadth of coverage at 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × sequencing depth obtained by COVseq vs. NEBNext for the same samples in ( e ). h Percentage of sequencing reads aligned to the SARS-CoV-2 reference genome, human reference genome (Hs), other genomes or unmapped, for the same samples in ( e ). The bottom plot shows the Ct value of each sample. i Correlation between the number of single-nucleotide variants (SNVs) per sample detected by COVseq (PE300) vs. NEBNext (SE75) in 20 ( n ) out of 29 OAS-29 samples with Ct ≤ 35. j Matrix showing the SNVs detected by COVseq, NEBNext, or both in the 20 OAS-29 samples with Ct ≤ 35. k Heatmap of the depth of coverage at the genomic positions of all the SNVs defining the UK (B.1.1.7), South African (B.1.351) and Brazilian (P.1) variants of concern (VOC) for the 20 OAS-29 samples with Ct ≤ 35 sequenced by COVseq. Gray color indicates locations that would have insufficient coverage to call SNVs (

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, High Throughput Screening Assay, Purification, Reverse Transcription Polymerase Chain Reaction

    9) Product Images from "The Glu727 Allele of Thyroid Stimulating Hormone Receptor Gene is Associated with Osteoporosis"

    Article Title: The Glu727 Allele of Thyroid Stimulating Hormone Receptor Gene is Associated with Osteoporosis

    Journal: North American Journal of Medical Sciences

    doi: 10.4103/1947-2714.98588

    Polymerase chain reaction (PCR) products at D727E site. PCR products were cut by NlaIII endonuclease and separated by agarose gel electrophoresis
    Figure Legend Snippet: Polymerase chain reaction (PCR) products at D727E site. PCR products were cut by NlaIII endonuclease and separated by agarose gel electrophoresis

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    10) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

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    New England Biolabs nlaiii
    Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) <t>SbfI-SphI</t> and ( B ) <t>PstI-NlaIII.</t>
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    Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Density plots of genomic relatedness (GR) among full siblings and non-siblings for: ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    Manhattan and quantile–quantile plots of the association tests for length and log 2 K in the PstI-NlaIII scenario ( n = 179). The red horizontal line indicates the Bonferroni error rate-adjusted significance level. The blue line indicates the threshold of the significant markers after BH adjustment of p -values.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Manhattan and quantile–quantile plots of the association tests for length and log 2 K in the PstI-NlaIII scenario ( n = 179). The red horizontal line indicates the Bonferroni error rate-adjusted significance level. The blue line indicates the threshold of the significant markers after BH adjustment of p -values.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    Distributions of post-filtering minor allele frequency (MAF) and single nucleotide polymorphism (SNP) call rate for SbfI-SphI ( n = 253) and the intersecting ( n = 175) animals that were also genotyped with PstI-NlaIII.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Distributions of post-filtering minor allele frequency (MAF) and single nucleotide polymorphism (SNP) call rate for SbfI-SphI ( n = 253) and the intersecting ( n = 175) animals that were also genotyped with PstI-NlaIII.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    Heatmaps visualizing the relative frequencies (%) of family predictions according to the DAPC cross-validation scheme for ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Heatmaps visualizing the relative frequencies (%) of family predictions according to the DAPC cross-validation scheme for ( A ) SbfI-SphI and ( B ) PstI-NlaIII.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    Discriminant analysis of principal components (DAPC) for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Discriminant analysis of principal components (DAPC) for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    Principal component analysis for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

    doi: 10.3390/ani11030899

    Figure Lengend Snippet: Principal component analysis for ( A ) SbfI-SphI and ( B ) PstI-NlaIII genotyping scenarios. The represented population is the intersection ( n = 175) of the individuals that were genotyped in both scenarios.

    Article Snippet: Briefly, each sample (15 ng/μL DNA) was digested at 37 °C for 60 min in the same reaction with either SbfI (recognizing the CCTGCA|GG motif) and SphI (recognizing the GCATG|C motif) or PstI (recognizing the CTGCA|G motif) and NlaIII (recognizing the CATG|N motif) high fidelity restriction enzymes (New England Biolabs, UK; NEB), by using 6 U of each enzyme per microgram of genomic DNA in 1× Reaction Buffer 4 (NEB).

    Techniques:

    V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .

    Journal: Cell

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning

    doi: 10.1016/j.cell.2018.04.035

    Figure Lengend Snippet: V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .

    Article Snippet: Nuclei were digested with 700 units of Nla III (NEB, #R0125) or Mse I (NEB, #R0525) restriction enzyme at 37°C overnight, followed by ligation under dilute conditions at 16°C overnight.

    Techniques: Polymerase Chain Reaction

    Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.

    Journal: Molecular Systems Biology

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

    doi: 10.15252/msb.20167311

    Figure Lengend Snippet: Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.

    Article Snippet: Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Techniques: Isolation, Ligation, In Situ, Chromatin Immunoprecipitation

    Native interactions are confined by TAD boundaries and describe prelooping i4C‐seq was performed in HUVECs using Nla III and the TSSs of BMP4 , CDKN3 , CNIH , and SAMD4A as viewpoints (triangles). Interactions are shown aligned to TAD boundaries (gray rectangles; from Dixon et al , 2012 ) and HUVEC ENCODE ChIP‐seq data (below). Prelooping of the SAMD4A and BMP4 TNF‐responsive TSSs to enhancers is indicated (orange lines).

    Journal: Molecular Systems Biology

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

    doi: 10.15252/msb.20167311

    Figure Lengend Snippet: Native interactions are confined by TAD boundaries and describe prelooping i4C‐seq was performed in HUVECs using Nla III and the TSSs of BMP4 , CDKN3 , CNIH , and SAMD4A as viewpoints (triangles). Interactions are shown aligned to TAD boundaries (gray rectangles; from Dixon et al , 2012 ) and HUVEC ENCODE ChIP‐seq data (below). Prelooping of the SAMD4A and BMP4 TNF‐responsive TSSs to enhancers is indicated (orange lines).

    Article Snippet: Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Techniques: Chromatin Immunoprecipitation

    Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations

    doi: 10.1128/JCM.40.5.1617-1620.2002

    Figure Lengend Snippet: Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.

    Article Snippet: The amplified 118-bp fragment was subjected to cleavage by Nla III (New England Biolabs) and Hae III (Amersham Pharmacia Biotech) restriction endonucleases, and the digests obtained were separated in 3% MetaPhor agarose gels (FMC BioProducts).

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis

    PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations

    doi: 10.1128/JCM.40.5.1617-1620.2002

    Figure Lengend Snippet: PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.

    Article Snippet: The amplified 118-bp fragment was subjected to cleavage by Nla III (New England Biolabs) and Hae III (Amersham Pharmacia Biotech) restriction endonucleases, and the digests obtained were separated in 3% MetaPhor agarose gels (FMC BioProducts).

    Techniques: Polymerase Chain Reaction, Amplification, Produced