a oralis  (Covaris)


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    Structured Review

    Covaris a oralis
    Phylogenetic tree highlighting position of Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces <t>urinae</t> strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces <t>oralis</t> strain Marseille-P3109 relative to other type strains within Actinomyces genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes sequences are indicated in brackets. Sequences were aligned using CLUSTALW ( http://www.clustal.org/clustal2/ ), and phylogenetic inferences were obtained using maximum-likelihood method within MEGA 6 ( http://www.megasoftware.net/mega.php ). Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1000 times to generate majority consensus tree. Actinobaculum urinale (NR 028978.1) was used as outgroup. Scale bar = 1% nucleotide sequence divergence.
    A Oralis, supplied by Covaris, used in various techniques. Bioz Stars score: 92/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Noncontiguous finished genome sequences and descriptions of Actinomyces ihuae, Actinomyces bouchesdurhonensis, Actinomyces urinae, Actinomyces marseillensis, Actinomyces mediterranea and Actinomyces oralis sp. nov. identified by culturomics"

    Article Title: Noncontiguous finished genome sequences and descriptions of Actinomyces ihuae, Actinomyces bouchesdurhonensis, Actinomyces urinae, Actinomyces marseillensis, Actinomyces mediterranea and Actinomyces oralis sp. nov. identified by culturomics

    Journal: New Microbes and New Infections

    doi: 10.1016/j.nmni.2018.06.004

    Phylogenetic tree highlighting position of Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109 relative to other type strains within Actinomyces genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes sequences are indicated in brackets. Sequences were aligned using CLUSTALW ( http://www.clustal.org/clustal2/ ), and phylogenetic inferences were obtained using maximum-likelihood method within MEGA 6 ( http://www.megasoftware.net/mega.php ). Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1000 times to generate majority consensus tree. Actinobaculum urinale (NR 028978.1) was used as outgroup. Scale bar = 1% nucleotide sequence divergence.
    Figure Legend Snippet: Phylogenetic tree highlighting position of Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109 relative to other type strains within Actinomyces genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes sequences are indicated in brackets. Sequences were aligned using CLUSTALW ( http://www.clustal.org/clustal2/ ), and phylogenetic inferences were obtained using maximum-likelihood method within MEGA 6 ( http://www.megasoftware.net/mega.php ). Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1000 times to generate majority consensus tree. Actinobaculum urinale (NR 028978.1) was used as outgroup. Scale bar = 1% nucleotide sequence divergence.

    Techniques Used: Sequencing

    Gram staining and electron micrographs, respectively, of Actinomyces oralis strain Marseille-P3109 (A, B), Actinomyces ihuae strain SD1 (C, D), Actinomyces bouchesdurhonensis strain Marseille-P2825 (E, F), Actinomyces urinae strain Marseille-P2225 (G, H), Actinomyces marseillensis strain Marseille-P2818 (I, J) and Actinomyces mediterranea strain Marseille-P3257 (K, L).
    Figure Legend Snippet: Gram staining and electron micrographs, respectively, of Actinomyces oralis strain Marseille-P3109 (A, B), Actinomyces ihuae strain SD1 (C, D), Actinomyces bouchesdurhonensis strain Marseille-P2825 (E, F), Actinomyces urinae strain Marseille-P2225 (G, H), Actinomyces marseillensis strain Marseille-P2818 (I, J) and Actinomyces mediterranea strain Marseille-P3257 (K, L).

    Techniques Used: Staining

    Reference mass spectra from Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109. Spectra from 12 individual colonies were compared and each reference spectrum generated (A). Gel view comparing Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109 to other species within genus Actinomyces . Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel–like look; x -axis indicates m/z value and left y -axis running spectrum number originating from subsequent spectra loading. Peak intensity expressed by greyscale scheme code. Colour bar and right y -axis indicate relation between colour peak, with peak intensity in arbitrary units. Displayed species are indicated at left (B).
    Figure Legend Snippet: Reference mass spectra from Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109. Spectra from 12 individual colonies were compared and each reference spectrum generated (A). Gel view comparing Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109 to other species within genus Actinomyces . Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel–like look; x -axis indicates m/z value and left y -axis running spectrum number originating from subsequent spectra loading. Peak intensity expressed by greyscale scheme code. Colour bar and right y -axis indicate relation between colour peak, with peak intensity in arbitrary units. Displayed species are indicated at left (B).

    Techniques Used: Generated

    2) Product Images from "Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing"

    Article Title: Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5381-7

    MinION reads improve assembly contiguity. The number of contigs (left), N50 (in Mbp, center), and assembly length (in Mbp, right) are shown for each of the MiSeq-based (SPAdes, Unicycler, SPAdes-hybrid, and Unicycler-hybrid) and MinION-based (Canu, Canu+Nanopolish, Canu+Pilon) genome assemblies. Results for Pseudonocardia , Aeromonas , and Flavobacterium are shown in blue, red, and green, respectively
    Figure Legend Snippet: MinION reads improve assembly contiguity. The number of contigs (left), N50 (in Mbp, center), and assembly length (in Mbp, right) are shown for each of the MiSeq-based (SPAdes, Unicycler, SPAdes-hybrid, and Unicycler-hybrid) and MinION-based (Canu, Canu+Nanopolish, Canu+Pilon) genome assemblies. Results for Pseudonocardia , Aeromonas , and Flavobacterium are shown in blue, red, and green, respectively

    Techniques Used:

    Comparison of Pseudonocardia assemblies generated during this study. (A): Heatmaps depicting Mash distances between the assemblies of each Pseudonocardia strain based on their shared k-mer content. Whiter colors indicate greater Mash distances between assemblies. (B): Mashtree analysis showing the relationships of all Pseudonocardia assemblies to each other, based on Mash distances. The scale bar represents a Mash distance of 0.003
    Figure Legend Snippet: Comparison of Pseudonocardia assemblies generated during this study. (A): Heatmaps depicting Mash distances between the assemblies of each Pseudonocardia strain based on their shared k-mer content. Whiter colors indicate greater Mash distances between assemblies. (B): Mashtree analysis showing the relationships of all Pseudonocardia assemblies to each other, based on Mash distances. The scale bar represents a Mash distance of 0.003

    Techniques Used: Generated

    Anvi’o analysis of annotation quality. Strains are grouped by species with Pseudonocardia shown in blue, Aeromonas shown in red, and Flavobacterium shown in green. Each heatmap row corresponds to an individual strain and each column corresponds to a unique assembly method
    Figure Legend Snippet: Anvi’o analysis of annotation quality. Strains are grouped by species with Pseudonocardia shown in blue, Aeromonas shown in red, and Flavobacterium shown in green. Each heatmap row corresponds to an individual strain and each column corresponds to a unique assembly method

    Techniques Used:

    3) Product Images from "Draft Genome Sequence of Pseudomonas sp. Strain MWU13-2860, Isolated from a Wild Cranberry Bog in Truro, Massachusetts"

    Article Title: Draft Genome Sequence of Pseudomonas sp. Strain MWU13-2860, Isolated from a Wild Cranberry Bog in Truro, Massachusetts

    Journal: Microbiology Resource Announcements

    doi: 10.1128/MRA.01007-18

    An evolutionary history (16S rRNA phylogeny) for Pseudomonas spp. commonly associated with soil and plant tissues, including isolate MWU13-2860, was inferred in MEGA7. Sequences were aligned by MUSCLE, and a maximum likelihood tree was constructed, with complete deletion of gaps and missing data, based on the Kimura 2-parameter model. The tree with the highest log likelihood (-4,713.97) is shown, with bootstrap values based on 500 iterations next to the branches. An initial tree was obtained by applying neighbor-joining and BioNJ algorithms to pairwise distances using the maximum composite likelihood (MCL) approach, followed by selecting the topology with a superior log-likelihood value. A discrete gamma distribution to model evolutionary rate differences among sites (+ G , parameter = 0.1370) and a rate variation model that allowed for some sites to be evolutionarily invariable ([+ I ], 60.10% of the sites) were used. Except for the Escherichia coli outgroup, the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A total of 1,320 positions were used in the final data set.
    Figure Legend Snippet: An evolutionary history (16S rRNA phylogeny) for Pseudomonas spp. commonly associated with soil and plant tissues, including isolate MWU13-2860, was inferred in MEGA7. Sequences were aligned by MUSCLE, and a maximum likelihood tree was constructed, with complete deletion of gaps and missing data, based on the Kimura 2-parameter model. The tree with the highest log likelihood (-4,713.97) is shown, with bootstrap values based on 500 iterations next to the branches. An initial tree was obtained by applying neighbor-joining and BioNJ algorithms to pairwise distances using the maximum composite likelihood (MCL) approach, followed by selecting the topology with a superior log-likelihood value. A discrete gamma distribution to model evolutionary rate differences among sites (+ G , parameter = 0.1370) and a rate variation model that allowed for some sites to be evolutionarily invariable ([+ I ], 60.10% of the sites) were used. Except for the Escherichia coli outgroup, the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A total of 1,320 positions were used in the final data set.

    Techniques Used: Construct

    4) Product Images from "Coordinate Regulation of DNA Methylation and H3K27me3 in Mouse Embryonic Stem Cells"

    Article Title: Coordinate Regulation of DNA Methylation and H3K27me3 in Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053880

    Global antagonism to H3K27me3 in Dnmt TKO cells. a, Classification of promoters identified in ChIP-seq experiment based on presence of H3K27me3 and H3K4me3 in wildtype cells. H3K4 and H3K27 methylation data from [5] . b, Profile of enrichment of ChIP-seq tags in 100 bp bins across the promoter for all genes with or without peaks of increased H3K27me3 in Dnmt TKO cells. c, Distribution of ChIP-seq reads according to genomic features. d, Number of ChIP-seq peaks intersecting with either fully-, low- or unmethylated regions according to data from [26] . e, Expression level of Eed in v6.5 and Dnmt TKO cells by qRT-PCR. f, Western blot analysis of EZH2 in v6.5 and Dnmt TKO cells. Relative intensity of EZH2 band from calculated using ImageJ is shown on the bottom. Intensity levels of EZH2 are normalized to Tubulin. h, Boxplot of expression level change for genes enriched in H3K27me3 in Dnmt TKO cells.
    Figure Legend Snippet: Global antagonism to H3K27me3 in Dnmt TKO cells. a, Classification of promoters identified in ChIP-seq experiment based on presence of H3K27me3 and H3K4me3 in wildtype cells. H3K4 and H3K27 methylation data from [5] . b, Profile of enrichment of ChIP-seq tags in 100 bp bins across the promoter for all genes with or without peaks of increased H3K27me3 in Dnmt TKO cells. c, Distribution of ChIP-seq reads according to genomic features. d, Number of ChIP-seq peaks intersecting with either fully-, low- or unmethylated regions according to data from [26] . e, Expression level of Eed in v6.5 and Dnmt TKO cells by qRT-PCR. f, Western blot analysis of EZH2 in v6.5 and Dnmt TKO cells. Relative intensity of EZH2 band from calculated using ImageJ is shown on the bottom. Intensity levels of EZH2 are normalized to Tubulin. h, Boxplot of expression level change for genes enriched in H3K27me3 in Dnmt TKO cells.

    Techniques Used: Chromatin Immunoprecipitation, Methylation, Expressing, Quantitative RT-PCR, Western Blot

    Increased H3K27me3 in Dnmt TKO ES cells. a, b, Average normalized fold enrichment of ChIP-seq reads in 100 bp bins in Dnmt TKO cells relative to wildtype for two representative loci, Tnp1 (a) and Prss21 (b). ChIP-seq peaks are indicated by the grey bars and genes are indicated at the bottom. c, qPCR validation of ChIP-seq peaks. Each bar represents the average percent input immunoprecipitated for six biological replicate ChIP experiments using chromatin from wild type v6.5 cells or Dnmt TKO . Note that all six ChIP experiments are independent of those used to generate the ChIP-seq libraries. Primers used in (c) are listed in Supplementary Table 4 (* p
    Figure Legend Snippet: Increased H3K27me3 in Dnmt TKO ES cells. a, b, Average normalized fold enrichment of ChIP-seq reads in 100 bp bins in Dnmt TKO cells relative to wildtype for two representative loci, Tnp1 (a) and Prss21 (b). ChIP-seq peaks are indicated by the grey bars and genes are indicated at the bottom. c, qPCR validation of ChIP-seq peaks. Each bar represents the average percent input immunoprecipitated for six biological replicate ChIP experiments using chromatin from wild type v6.5 cells or Dnmt TKO . Note that all six ChIP experiments are independent of those used to generate the ChIP-seq libraries. Primers used in (c) are listed in Supplementary Table 4 (* p

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation

    5) Product Images from "Comparative Genomics of a Plant-Pathogenic Fungus, Pyrenophora tritici-repentis, Reveals Transduplication and the Impact of Repeat Elements on Pathogenicity and Population Divergence"

    Article Title: Comparative Genomics of a Plant-Pathogenic Fungus, Pyrenophora tritici-repentis, Reveals Transduplication and the Impact of Repeat Elements on Pathogenicity and Population Divergence

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.112.004044

    Predicted secreted proteins in the genome sequence of Pyrenophora tritici-repentis . (A) Distribution of secreted proteins in the reference Ptr isolate (BFP-ToxAC) and the Illumina-sequenced pathogenic (DW7-ToxB) and nonpathogenic (SD20-NP) isolates. (B) PCR screen of pathogenic and nonpathogenic isolates for the presence of the gene and transcript for PTRG_11888, a predicted putative secreted protein. Pathogenic isolates: BFP-ToxAC; ASC1; 86-124; D308; DW2; SO3-P; Non-pathogenic isolates: SD20-NP; 90-2; 98-31-2 (see also Table S1 ).
    Figure Legend Snippet: Predicted secreted proteins in the genome sequence of Pyrenophora tritici-repentis . (A) Distribution of secreted proteins in the reference Ptr isolate (BFP-ToxAC) and the Illumina-sequenced pathogenic (DW7-ToxB) and nonpathogenic (SD20-NP) isolates. (B) PCR screen of pathogenic and nonpathogenic isolates for the presence of the gene and transcript for PTRG_11888, a predicted putative secreted protein. Pathogenic isolates: BFP-ToxAC; ASC1; 86-124; D308; DW2; SO3-P; Non-pathogenic isolates: SD20-NP; 90-2; 98-31-2 (see also Table S1 ).

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Mapping of sequence reads of resequenced isolates relative to the reference genome of Pyrenophora tritici-repentis . Schematic represents the reference genome scaffolds (gray bars with supercontig numbers) mapped to each chromosome as defined by the reference optical map. The border box reflects chromosome size as indicated next to the chromosome number and white spaces between the scaffolds represent gaps in the sequ ence assemblies. Repeat density (green) in the reference isolate, and read (gray central panel) and SNP (top panel) densities of the Illumina-sequenced isolates (DW7-ToxB-red, SD20-NP-blue), were mapped per 10 kb of the high-quality genome assembly based on the reference isolate (BFP-ToxAC) of P. tritici-repentis .
    Figure Legend Snippet: Mapping of sequence reads of resequenced isolates relative to the reference genome of Pyrenophora tritici-repentis . Schematic represents the reference genome scaffolds (gray bars with supercontig numbers) mapped to each chromosome as defined by the reference optical map. The border box reflects chromosome size as indicated next to the chromosome number and white spaces between the scaffolds represent gaps in the sequ ence assemblies. Repeat density (green) in the reference isolate, and read (gray central panel) and SNP (top panel) densities of the Illumina-sequenced isolates (DW7-ToxB-red, SD20-NP-blue), were mapped per 10 kb of the high-quality genome assembly based on the reference isolate (BFP-ToxAC) of P. tritici-repentis .

    Techniques Used: Sequencing

    Absence of the 145-kb ToxA -containing region in the pathogenic DW7-ToxB and non-pathogenic SD20-NP isolates of Pyrenophora tritici-repentis . Schematic includes the protein-coding regions (purple) and repeats (green) present within the 170-kb ToxA -containing genomic region in the BFP-ToxAC reference genome. The graphs show coverage of reads obtained from the pathogenic DW7-ToxB and nonpathogenic SD20-NP isolates. Coverage depth per nucleotide (nt) is indicated on the left and fold coverage is indicated on the right. Blue shading represents synteny between genomic scaffolds of Cochliobolus heterostrophus isolate C4, SD20-NP, DW7-ToxB, and flanking regions of the 170-kb ToxA -containing region of the reference. Similarly colored bars within the scaffolds indicate colinear blocks.
    Figure Legend Snippet: Absence of the 145-kb ToxA -containing region in the pathogenic DW7-ToxB and non-pathogenic SD20-NP isolates of Pyrenophora tritici-repentis . Schematic includes the protein-coding regions (purple) and repeats (green) present within the 170-kb ToxA -containing genomic region in the BFP-ToxAC reference genome. The graphs show coverage of reads obtained from the pathogenic DW7-ToxB and nonpathogenic SD20-NP isolates. Coverage depth per nucleotide (nt) is indicated on the left and fold coverage is indicated on the right. Blue shading represents synteny between genomic scaffolds of Cochliobolus heterostrophus isolate C4, SD20-NP, DW7-ToxB, and flanking regions of the 170-kb ToxA -containing region of the reference. Similarly colored bars within the scaffolds indicate colinear blocks.

    Techniques Used:

    Phylogeny of Pyrenophora tritici-repentis and symptoms induced by the three sequenced isolates. RAxML maximum likelihood phylogenetic tree (best tree -nL1505566.555282) inferred from a 100-protein superalignment comprising 93,210 amino acid positions (A). Subphyla (-mycotina) and classes (-mycetes) of the phylum Ascomycota are shown and numbers near nodes are bootstrap partitions. (B) ToxA and ToxC symptoms induced by BFP-ToxAC (on Glenlea and 6B365, respectively; top 2 leaves), ToxB symptoms induced by DW7-ToxB (on 6B662; middle leaf), and the resistant reaction produced by the nonpathogenic SD20-NP (on Auburn; bottom leaf).
    Figure Legend Snippet: Phylogeny of Pyrenophora tritici-repentis and symptoms induced by the three sequenced isolates. RAxML maximum likelihood phylogenetic tree (best tree -nL1505566.555282) inferred from a 100-protein superalignment comprising 93,210 amino acid positions (A). Subphyla (-mycotina) and classes (-mycetes) of the phylum Ascomycota are shown and numbers near nodes are bootstrap partitions. (B) ToxA and ToxC symptoms induced by BFP-ToxAC (on Glenlea and 6B365, respectively; top 2 leaves), ToxB symptoms induced by DW7-ToxB (on 6B662; middle leaf), and the resistant reaction produced by the nonpathogenic SD20-NP (on Auburn; bottom leaf).

    Techniques Used: Produced

    Read mapping to and de novo assembly of ToxB - and toxb -containing loci in the genome of Pyrenophora tritici-repentis . Schematic of Illumina sequence reads (line graph) of isolate DW7-ToxB mapped to the ToxB1 locus (top: ToxB1 locus; accession number: AY425480.1) and of SD20-NP mapped to the toxb locus (bottom: toxb locus; accession number: AY083456.2). Coverage depth per nt is indicated on the left and fold coverage is indicated on the right. Straight lines above the graphs depict the contigs present in the de novo assemblies of the Illumina-sequenced isolates. The arrow on the contig above the toxb locus shows how that contig extends beyond the locus.
    Figure Legend Snippet: Read mapping to and de novo assembly of ToxB - and toxb -containing loci in the genome of Pyrenophora tritici-repentis . Schematic of Illumina sequence reads (line graph) of isolate DW7-ToxB mapped to the ToxB1 locus (top: ToxB1 locus; accession number: AY425480.1) and of SD20-NP mapped to the toxb locus (bottom: toxb locus; accession number: AY083456.2). Coverage depth per nt is indicated on the left and fold coverage is indicated on the right. Straight lines above the graphs depict the contigs present in the de novo assemblies of the Illumina-sequenced isolates. The arrow on the contig above the toxb locus shows how that contig extends beyond the locus.

    Techniques Used: Sequencing

    Transduplication of a histone H3-like protein family in the genome of Pyrenophora tritici-repentis . (A) Two representatives of a transduplicating family of DNA transposable elements in the BFP-ToxAC reference genome are shown with the characteristic motifs of the elements indicated (brown box = terminal inverted repeats (TIR); green box = hAT transposon coding regions; purple box = hAT dimerization motif; blue box = central open reading frame (ORF) coding region; yellow box = histone H3 coding region; black boundary box = full-length element). The large, likely autonomous element is approximately 5.6 kb in length. Black lines between elements illustrate a pathogen-specific recombination event that results in a smaller element of approximately 2.3 kb. The graphs show coverage of reads obtained from the pathogenic DW7-ToxB and nonpathogenic SD20-NP isolates. Arrows and lines below the element representation indicate alignments of ESTs detected in various libraries (arrows = poly(A) tails, light gray = introns). (B) Alignment of novel histone H3-like (H3L) proteins identified in Ptr with bona fide histone H3 variants from Ptr , S. nodorum , and N. crassa . Four variants of full-length H3L exist in Ptr , all are different by a single aa change (underlined; also see Table S9 ). Two changes in H3L alleles result in frameshift mutations, yielding 5′ and 3′ truncated versions of H3L. Most (21) copies are identical to PTRG_00559.1 in amino acid and DNA sequence. Completely conserved residues are shown in black, similar residues in green and variable residues in gray.
    Figure Legend Snippet: Transduplication of a histone H3-like protein family in the genome of Pyrenophora tritici-repentis . (A) Two representatives of a transduplicating family of DNA transposable elements in the BFP-ToxAC reference genome are shown with the characteristic motifs of the elements indicated (brown box = terminal inverted repeats (TIR); green box = hAT transposon coding regions; purple box = hAT dimerization motif; blue box = central open reading frame (ORF) coding region; yellow box = histone H3 coding region; black boundary box = full-length element). The large, likely autonomous element is approximately 5.6 kb in length. Black lines between elements illustrate a pathogen-specific recombination event that results in a smaller element of approximately 2.3 kb. The graphs show coverage of reads obtained from the pathogenic DW7-ToxB and nonpathogenic SD20-NP isolates. Arrows and lines below the element representation indicate alignments of ESTs detected in various libraries (arrows = poly(A) tails, light gray = introns). (B) Alignment of novel histone H3-like (H3L) proteins identified in Ptr with bona fide histone H3 variants from Ptr , S. nodorum , and N. crassa . Four variants of full-length H3L exist in Ptr , all are different by a single aa change (underlined; also see Table S9 ). Two changes in H3L alleles result in frameshift mutations, yielding 5′ and 3′ truncated versions of H3L. Most (21) copies are identical to PTRG_00559.1 in amino acid and DNA sequence. Completely conserved residues are shown in black, similar residues in green and variable residues in gray.

    Techniques Used: HAT Assay, Sequencing

    6) Product Images from "Single-cell paired-end genome sequencing reveals structural variation per cell cycle"

    Article Title: Single-cell paired-end genome sequencing reveals structural variation per cell cycle

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt345

    Accuracy of WGA nucleotide copying and genotyping. ( A ) Nucleotide mismatch frequency with the hg19-reference genome at each base of the read. Only bases with a base-call quality of ≥30 in reads having a minimum mapping quality of 30 were considered. It is clear that the PicoPlex WGA method introduces significantly more WGA nucleotide errors than MDA. ( B and C ) Approximately 450 000 SNPs, which were heterozygous in the sequences of two HCC38 subclones (B8FF4C and B8FB3A), were genotyped in the single-cell sequences. (B) Single-cell SNP zygosity concordance with the reference genotype ( y -axis) in function of read depth across the SNPs ( x -axis). (C) Single-cell SNP call-rate ( y -axis) in function of read depth across the SNPs ( x -axis).
    Figure Legend Snippet: Accuracy of WGA nucleotide copying and genotyping. ( A ) Nucleotide mismatch frequency with the hg19-reference genome at each base of the read. Only bases with a base-call quality of ≥30 in reads having a minimum mapping quality of 30 were considered. It is clear that the PicoPlex WGA method introduces significantly more WGA nucleotide errors than MDA. ( B and C ) Approximately 450 000 SNPs, which were heterozygous in the sequences of two HCC38 subclones (B8FF4C and B8FB3A), were genotyped in the single-cell sequences. (B) Single-cell SNP zygosity concordance with the reference genotype ( y -axis) in function of read depth across the SNPs ( x -axis). (C) Single-cell SNP call-rate ( y -axis) in function of read depth across the SNPs ( x -axis).

    Techniques Used: Whole Genome Amplification, Multiple Displacement Amplification

    Single-cell DNA copy number profiling by focal read-depth analysis. ( A ) A tree of the single-cell–derived subclones and isolated HCC38-tumour cells. ( B ) Concordances of the DNA copy number profiles of the MDA-WGAed cells (blue), the PicoPlex-WGAed cells (red) and the non-WGAed subclones (green) with the reference B8FF4C copy number profile. The copy number concordance between a sample and B8FF4C was calculated by comparing the copy number states of each 10-kb bin genome wide following focal sequence-depth analyses. The y -axis represents the copy number concordance, the x -axis the γ penalty parameter of the PCF algorithm used for segmentation (‘Materials and Methods’ section). The mean copy number concordance is depicted as a line, the standard deviation as a shaded region. Two vertical dashed lines indicate the γ values of 25 and 150, respectively. ( C ) Complementary DNA copy number changes on chromosome 5 in two sister cells related by one cell cycle. Orange lines, representing the B8FF4C copy number segments, are overlaid on top of the red lines, which represent the single-cell PicoPlex copy number segments. (Top) Cell ‘PicoPlex-sc9’, (bottom) cell ‘PicoPlex-sc10’. ( D ) Segments of integer DNA copy number states following focal sequence-depth analyses using 10-kb bins and PCF segmentation (γ = 25) across all autosomes and the X chromosome. The integer DNA copy number is depicted as a heat map of which a color legend has been integrated in the figure. The profiles of the non-WGA single-cell–derived subclone samples (A6GD7A, A6GE4F and B8FB3A) and the reference B8FF4C sample are shown, followed by the four PicoPlex-amplified single cells (PicoPlex-sc1, PicoPlex-sc2, PicoPlex-sc9 and PicoPlex-sc10) and the four MDA-amplified single cells (mda-sc82, mda-sc83, mda-sc1 and mda-sc2).
    Figure Legend Snippet: Single-cell DNA copy number profiling by focal read-depth analysis. ( A ) A tree of the single-cell–derived subclones and isolated HCC38-tumour cells. ( B ) Concordances of the DNA copy number profiles of the MDA-WGAed cells (blue), the PicoPlex-WGAed cells (red) and the non-WGAed subclones (green) with the reference B8FF4C copy number profile. The copy number concordance between a sample and B8FF4C was calculated by comparing the copy number states of each 10-kb bin genome wide following focal sequence-depth analyses. The y -axis represents the copy number concordance, the x -axis the γ penalty parameter of the PCF algorithm used for segmentation (‘Materials and Methods’ section). The mean copy number concordance is depicted as a line, the standard deviation as a shaded region. Two vertical dashed lines indicate the γ values of 25 and 150, respectively. ( C ) Complementary DNA copy number changes on chromosome 5 in two sister cells related by one cell cycle. Orange lines, representing the B8FF4C copy number segments, are overlaid on top of the red lines, which represent the single-cell PicoPlex copy number segments. (Top) Cell ‘PicoPlex-sc9’, (bottom) cell ‘PicoPlex-sc10’. ( D ) Segments of integer DNA copy number states following focal sequence-depth analyses using 10-kb bins and PCF segmentation (γ = 25) across all autosomes and the X chromosome. The integer DNA copy number is depicted as a heat map of which a color legend has been integrated in the figure. The profiles of the non-WGA single-cell–derived subclone samples (A6GD7A, A6GE4F and B8FB3A) and the reference B8FF4C sample are shown, followed by the four PicoPlex-amplified single cells (PicoPlex-sc1, PicoPlex-sc2, PicoPlex-sc9 and PicoPlex-sc10) and the four MDA-amplified single cells (mda-sc82, mda-sc83, mda-sc1 and mda-sc2).

    Techniques Used: Derivative Assay, Isolation, Multiple Displacement Amplification, Genome Wide, Sequencing, Standard Deviation, Whole Genome Amplification, Amplification

    Sensitivity and positive predictive value of single-cell paired-end maps. ( A ) Sensitivity of the single-cell paired-end maps in function of thresholds on the minimum amount of discordant read pairs that had to support a rearrangement signature. A set of 24 deletion-, 124 tandem duplication-, 18 inversion- and 31 inter-chromosomal signatures confirmed by PCR in HCC38 were scored for their presence in the single-cell paired-end maps. The mean sensitivity across the non-WGA subclone, the single-cell MDA and the single-cell PicoPlex paired-end maps are depicted in the y -axis. Sensitivities for deletion, tandem duplication, inter-chromosomal rearrangement and inversion signatures are shown separately. For the computations, the refined paired-end maps were used that contained only rearrangement signatures supported by a minimum threshold amount of read pairs (= x -axis). ( B ) Positive predictive values of the single-cell paired-end maps for deletion, tandem duplication, inter-chromosomal rearrangement and inversion signatures in function of thresholds on the minimum amount of discordant read pairs that had to support a rearrangement signature. The positive predictive values (= y -axis) were computed as the amount of single-cell rearrangements with a matching rearrangement signature in the reference B8FF4C paired-end map, divided by the total number of single-cell rearrangements present in the respective single-cell paired-end map. Refined paired-end maps that contained only rearrangement signatures supported by a minimum threshold-amount of discordant read pairs (= x -axis) and which encompassed > 5 kb (except for putative inter-chromosomal events) were used for all calculations. The reference B8FF4C paired-end map consisted of signatures that encompassed > 5 kb (except for putative inter-chromosomal events) and that were supported by two or more discordantly mapping read pairs. ( C ) A Circos-plot depicting confirmed HCC38 rearrangements identified in single cell ‘mda-sc82’ following paired-end sequencing of the MDA product. From the outside to the inside of the Circos-plot: (i) chromosome ideograms, (ii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the non-WGA B8FF4C subclone, (iii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the single-cell ‘mda-sc82’ sample, (iv) the amount of read pairs supporting each single-cell rearrangement is depicted by a bar (scale 2–30) at the start of each rearrangement signature and (v) confirmed HCC38 rearrangements identified in single cell ‘mda-sc82’ following paired-end sequencing. Color legends for the rearrangements and the copy number heat map are indicated. ( D ) A Circos-plot depicting confirmed HCC38 rearrangements identified in single cell ‘PicoPlex-sc2’ following paired-end sequencing. From the outside to the inside of the Circos-plot: (i) chromosome ideograms, (ii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the non-WGA B8FF4C subclone, (iii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the single-cell ‘PicoPlex-sc2’ sample, (iv) the amount of read pairs supporting each single-cell rearrangement is depicted by a bar (scale 2–30) at the start of each rearrangement signature and (v) confirmed HCC38 rearrangements identified in single cell ‘PicoPlex-sc2’ following paired-end sequencing. Color legends for the rearrangements and the copy number heat map are indicated. Circos-plots depicting confirmed HCC38 rearrangements that are identified in all non-WGA subclone and single-cell paired-end maps individually are presented in Supplementary Figure S8 .
    Figure Legend Snippet: Sensitivity and positive predictive value of single-cell paired-end maps. ( A ) Sensitivity of the single-cell paired-end maps in function of thresholds on the minimum amount of discordant read pairs that had to support a rearrangement signature. A set of 24 deletion-, 124 tandem duplication-, 18 inversion- and 31 inter-chromosomal signatures confirmed by PCR in HCC38 were scored for their presence in the single-cell paired-end maps. The mean sensitivity across the non-WGA subclone, the single-cell MDA and the single-cell PicoPlex paired-end maps are depicted in the y -axis. Sensitivities for deletion, tandem duplication, inter-chromosomal rearrangement and inversion signatures are shown separately. For the computations, the refined paired-end maps were used that contained only rearrangement signatures supported by a minimum threshold amount of read pairs (= x -axis). ( B ) Positive predictive values of the single-cell paired-end maps for deletion, tandem duplication, inter-chromosomal rearrangement and inversion signatures in function of thresholds on the minimum amount of discordant read pairs that had to support a rearrangement signature. The positive predictive values (= y -axis) were computed as the amount of single-cell rearrangements with a matching rearrangement signature in the reference B8FF4C paired-end map, divided by the total number of single-cell rearrangements present in the respective single-cell paired-end map. Refined paired-end maps that contained only rearrangement signatures supported by a minimum threshold-amount of discordant read pairs (= x -axis) and which encompassed > 5 kb (except for putative inter-chromosomal events) were used for all calculations. The reference B8FF4C paired-end map consisted of signatures that encompassed > 5 kb (except for putative inter-chromosomal events) and that were supported by two or more discordantly mapping read pairs. ( C ) A Circos-plot depicting confirmed HCC38 rearrangements identified in single cell ‘mda-sc82’ following paired-end sequencing of the MDA product. From the outside to the inside of the Circos-plot: (i) chromosome ideograms, (ii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the non-WGA B8FF4C subclone, (iii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the single-cell ‘mda-sc82’ sample, (iv) the amount of read pairs supporting each single-cell rearrangement is depicted by a bar (scale 2–30) at the start of each rearrangement signature and (v) confirmed HCC38 rearrangements identified in single cell ‘mda-sc82’ following paired-end sequencing. Color legends for the rearrangements and the copy number heat map are indicated. ( D ) A Circos-plot depicting confirmed HCC38 rearrangements identified in single cell ‘PicoPlex-sc2’ following paired-end sequencing. From the outside to the inside of the Circos-plot: (i) chromosome ideograms, (ii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the non-WGA B8FF4C subclone, (iii) the integer DNA copy number heat map (using 10-kb bins and γ = 25) of the single-cell ‘PicoPlex-sc2’ sample, (iv) the amount of read pairs supporting each single-cell rearrangement is depicted by a bar (scale 2–30) at the start of each rearrangement signature and (v) confirmed HCC38 rearrangements identified in single cell ‘PicoPlex-sc2’ following paired-end sequencing. Color legends for the rearrangements and the copy number heat map are indicated. Circos-plots depicting confirmed HCC38 rearrangements that are identified in all non-WGA subclone and single-cell paired-end maps individually are presented in Supplementary Figure S8 .

    Techniques Used: Polymerase Chain Reaction, Whole Genome Amplification, Multiple Displacement Amplification, Sequencing

    7) Product Images from "Metagenomic and Metatranscriptomic Analysis of Microbial Community Structure and Gene Expression of Activated Sludge"

    Article Title: Metagenomic and Metatranscriptomic Analysis of Microbial Community Structure and Gene Expression of Activated Sludge

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038183

    Combined taxonomic domain information of DNA and cDNA datasets. Total DNA sequences and cDNA sequences were assigned to Bacteria , Eukaryota , Archaea , viruses, and other sequences.
    Figure Legend Snippet: Combined taxonomic domain information of DNA and cDNA datasets. Total DNA sequences and cDNA sequences were assigned to Bacteria , Eukaryota , Archaea , viruses, and other sequences.

    Techniques Used:

    8) Product Images from "Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis"

    Article Title: Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

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

    doi: 10.1093/dnares/dsv007

    Colonial characteristic and microscopic morphology of Bipolaris papendorfii UM 226. The surface (A) and reverse (B) colony morphology of B. papendorfii UM 226 after being cultured for 7 days. Light micrograph showing (C) typical zig-zag conidiophore with several conidia and (D) conidia with three pseudoseptates (×400 magnification, bars 20 µm). Scanning electron micrograph showing (E) zig-zag conidiophores with verruculose walled conidia (×3,065 magnification, bars 10 µm). This figure is available in black and white in print and in colour at DNA Research online.
    Figure Legend Snippet: Colonial characteristic and microscopic morphology of Bipolaris papendorfii UM 226. The surface (A) and reverse (B) colony morphology of B. papendorfii UM 226 after being cultured for 7 days. Light micrograph showing (C) typical zig-zag conidiophore with several conidia and (D) conidia with three pseudoseptates (×400 magnification, bars 20 µm). Scanning electron micrograph showing (E) zig-zag conidiophores with verruculose walled conidia (×3,065 magnification, bars 10 µm). This figure is available in black and white in print and in colour at DNA Research online.

    Techniques Used: Cell Culture

    MAT1-2 gene of Bipolaris papendorfii UM 226. (A) Schematic representation of major open reading frames (ORFs) identified in the MAT regions of B. papendorfii UM 226 and Cochliobolus heterostrophus C4. Numbers are in kilobases. (B) Bayesian phylogram generated based on MAT1-2 nucleotide sequences. The tree is rooted with Alternaria alternata as outgroup. Numbers on the nodes indicate Bayesian posterior probability based on 100 sampling frequency for a total of 150,000 generations. This figure is available in black and white in print and in colour at DNA Research online.
    Figure Legend Snippet: MAT1-2 gene of Bipolaris papendorfii UM 226. (A) Schematic representation of major open reading frames (ORFs) identified in the MAT regions of B. papendorfii UM 226 and Cochliobolus heterostrophus C4. Numbers are in kilobases. (B) Bayesian phylogram generated based on MAT1-2 nucleotide sequences. The tree is rooted with Alternaria alternata as outgroup. Numbers on the nodes indicate Bayesian posterior probability based on 100 sampling frequency for a total of 150,000 generations. This figure is available in black and white in print and in colour at DNA Research online.

    Techniques Used: Generated, Sampling

    9) Product Images from "Ubiquitous L1 Mosaicism in Hippocampal Neurons"

    Article Title: Ubiquitous L1 Mosaicism in Hippocampal Neurons

    Journal: Cell

    doi: 10.1016/j.cell.2015.03.026

    Single-Cell RC-Seq Workflow (A) NeuN + hippocampal nuclei were first purified by FACS (see also Figure S1 ). (B) Nuclei were then picked using a self-contained microscope and micromanipulator. (C) DNA was extracted from nuclei and subjected to linear WGA, followed by exponential PCR in two separate reactions for each nucleus, using different enzymes. (D) Exponential WGA products for each nucleus were combined, used to prepare Illumina libraries, and analyzed via WGS to assess genome coverage and possible amplification biases. (E) Libraries prepared in (D) were enriched via hybridization to L1-Ta LNA probes. (F) Enriched libraries were sequenced with 2 × 150-mer Illumina reads and analyzed to identify novel L1 integration sites (see also Figure S2 ).
    Figure Legend Snippet: Single-Cell RC-Seq Workflow (A) NeuN + hippocampal nuclei were first purified by FACS (see also Figure S1 ). (B) Nuclei were then picked using a self-contained microscope and micromanipulator. (C) DNA was extracted from nuclei and subjected to linear WGA, followed by exponential PCR in two separate reactions for each nucleus, using different enzymes. (D) Exponential WGA products for each nucleus were combined, used to prepare Illumina libraries, and analyzed via WGS to assess genome coverage and possible amplification biases. (E) Libraries prepared in (D) were enriched via hybridization to L1-Ta LNA probes. (F) Enriched libraries were sequenced with 2 × 150-mer Illumina reads and analyzed to identify novel L1 integration sites (see also Figure S2 ).

    Techniques Used: Purification, FACS, Microscopy, Whole Genome Amplification, Polymerase Chain Reaction, Amplification, Hybridization

    10) Product Images from "Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses"

    Article Title: Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2103.141169

    Diagnostic algorithm for identification of an unknown risk for influenza by using nanomicroarray and next-generation sequencing (NGS) assays. To determine the virus type for a suspected influenza virus infection, viral RNA is extracted from a patient sample and initially analyzed in nanomicroarray assay for screening and determining the influenza A and B viruses (1). Once a novel, emerging, or co-infected influenza A and B virus is found, universal reverse transcription PCR (RT-PCR) is performed to generate whole-genome mega-amplicons (2), which can then be retested on the nanomicroarray assay to confirm the initial finding (3) or sent to the central laboratory performing the NGS assay and data analysis for final sequence confirmation (4).
    Figure Legend Snippet: Diagnostic algorithm for identification of an unknown risk for influenza by using nanomicroarray and next-generation sequencing (NGS) assays. To determine the virus type for a suspected influenza virus infection, viral RNA is extracted from a patient sample and initially analyzed in nanomicroarray assay for screening and determining the influenza A and B viruses (1). Once a novel, emerging, or co-infected influenza A and B virus is found, universal reverse transcription PCR (RT-PCR) is performed to generate whole-genome mega-amplicons (2), which can then be retested on the nanomicroarray assay to confirm the initial finding (3) or sent to the central laboratory performing the NGS assay and data analysis for final sequence confirmation (4).

    Techniques Used: Diagnostic Assay, Next-Generation Sequencing, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    11) Product Images from "Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses"

    Article Title: Nanomicroarray and Multiplex Next-Generation Sequencing for Simultaneous Identification and Characterization of Influenza Viruses

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2103.141169

    Diagnostic algorithm for identification of an unknown risk for influenza by using nanomicroarray and next-generation sequencing (NGS) assays. To determine the virus type for a suspected influenza virus infection, viral RNA is extracted from a patient sample and initially analyzed in nanomicroarray assay for screening and determining the influenza A and B viruses (1). Once a novel, emerging, or co-infected influenza A and B virus is found, universal reverse transcription PCR (RT-PCR) is performed to generate whole-genome mega-amplicons (2), which can then be retested on the nanomicroarray assay to confirm the initial finding (3) or sent to the central laboratory performing the NGS assay and data analysis for final sequence confirmation (4).
    Figure Legend Snippet: Diagnostic algorithm for identification of an unknown risk for influenza by using nanomicroarray and next-generation sequencing (NGS) assays. To determine the virus type for a suspected influenza virus infection, viral RNA is extracted from a patient sample and initially analyzed in nanomicroarray assay for screening and determining the influenza A and B viruses (1). Once a novel, emerging, or co-infected influenza A and B virus is found, universal reverse transcription PCR (RT-PCR) is performed to generate whole-genome mega-amplicons (2), which can then be retested on the nanomicroarray assay to confirm the initial finding (3) or sent to the central laboratory performing the NGS assay and data analysis for final sequence confirmation (4).

    Techniques Used: Diagnostic Assay, Next-Generation Sequencing, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    12) Product Images from "Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes"

    Article Title: Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-1

    A plot of genome coverage against normalized average depth . Duplicate data sets were normalized and pooled. Variance in coverage above and below the normalized average depth (red vertical line) across the genome is shown. Deviation of sample curves from the average depth indicates level of evenness in coverage depth distribution across the genome. The closer the sample curve is to the vertical line, the more even the coverage. The theoretical curve represents average normalized depth at 100% genome coverage. A) Coverage by libraries made from P. falciparum 3D7 (1 normalized depth represents 21×). B) Coverage by libraries made from clinical isolate, PK0076 (1 normalized depth represents 11×). Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC.
    Figure Legend Snippet: A plot of genome coverage against normalized average depth . Duplicate data sets were normalized and pooled. Variance in coverage above and below the normalized average depth (red vertical line) across the genome is shown. Deviation of sample curves from the average depth indicates level of evenness in coverage depth distribution across the genome. The closer the sample curve is to the vertical line, the more even the coverage. The theoretical curve represents average normalized depth at 100% genome coverage. A) Coverage by libraries made from P. falciparum 3D7 (1 normalized depth represents 21×). B) Coverage by libraries made from clinical isolate, PK0076 (1 normalized depth represents 11×). Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC.

    Techniques Used:

    GC profile analysis of sequenced data . The GC content distribution for different library preparation methods are shown alongside theoretical data for comparison. A) GC content analysis on libraries prepared from P. falciparum 3D7 with mapped reads normalized to 21× genome coverage. B) GC content of libraries prepared from a clinical isolate (PK0076) with mapped reads normalized to 11× genome coverage. Libraries with GC content above 19.4% (the GC content of the P. falciparum 3D7 reference genome) indicate amplification bias towards templates with neutral GC composition. C) Artemis [ 9 , 10 ] screen view of coverage (mapped reads normalized to 21× genome coverage) for a PCR-free library and four other libraries under test on P. falciparum 3D7 chromosome 1 (zoomed in to show coverage on the GC rich telomere). Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. See additional file 1 , Figure S2 A B for coverage on the entire chromosome 1 and AT-rich locus.
    Figure Legend Snippet: GC profile analysis of sequenced data . The GC content distribution for different library preparation methods are shown alongside theoretical data for comparison. A) GC content analysis on libraries prepared from P. falciparum 3D7 with mapped reads normalized to 21× genome coverage. B) GC content of libraries prepared from a clinical isolate (PK0076) with mapped reads normalized to 11× genome coverage. Libraries with GC content above 19.4% (the GC content of the P. falciparum 3D7 reference genome) indicate amplification bias towards templates with neutral GC composition. C) Artemis [ 9 , 10 ] screen view of coverage (mapped reads normalized to 21× genome coverage) for a PCR-free library and four other libraries under test on P. falciparum 3D7 chromosome 1 (zoomed in to show coverage on the GC rich telomere). Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. See additional file 1 , Figure S2 A B for coverage on the entire chromosome 1 and AT-rich locus.

    Techniques Used: Amplification, Polymerase Chain Reaction

    Box plots showing coverage analysis of P. falciparum chromosome 11 . (i) P. falciparum 3D7; mapped reads normalized to 21× genome coverage (1 normalized depth represents 21×). (ii) Clinical isolate PK0076; mapped reads normalized to 11× genome coverage (1 normalized depth represents 11×). Subplots B, C and D in both i ii show coverage of sub-regions of the P. falciparum 3D7 chromosome 11. A) Coverage depth variability plotted for each library on the entire chromosome. B) Distribution of base coverage depth for each library over gene Pf11_0074 and its neighboring introns. C) Distribution of base coverage depth at positions 259985-260864 (extreme AT-region). D) Distribution of base coverage depth at positions 29092-30361 (VAR gene and introns). Top and bottom sides of a box plot represent 75 th and 25 th percentile of base coverage-depth distribution respectively. The middle line represents 50 th percentile. A narrow box indicates less variation in coverage depth across that locus and vice versa. Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. All P. falciparum 3D7and most clinical isolate libraries were prepared in duplicate and each replicate data plotted independently as shown.
    Figure Legend Snippet: Box plots showing coverage analysis of P. falciparum chromosome 11 . (i) P. falciparum 3D7; mapped reads normalized to 21× genome coverage (1 normalized depth represents 21×). (ii) Clinical isolate PK0076; mapped reads normalized to 11× genome coverage (1 normalized depth represents 11×). Subplots B, C and D in both i ii show coverage of sub-regions of the P. falciparum 3D7 chromosome 11. A) Coverage depth variability plotted for each library on the entire chromosome. B) Distribution of base coverage depth for each library over gene Pf11_0074 and its neighboring introns. C) Distribution of base coverage depth at positions 259985-260864 (extreme AT-region). D) Distribution of base coverage depth at positions 29092-30361 (VAR gene and introns). Top and bottom sides of a box plot represent 75 th and 25 th percentile of base coverage-depth distribution respectively. The middle line represents 50 th percentile. A narrow box indicates less variation in coverage depth across that locus and vice versa. Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. All P. falciparum 3D7and most clinical isolate libraries were prepared in duplicate and each replicate data plotted independently as shown.

    Techniques Used:

    13) Product Images from "Ultra-precise detection of mutations by droplet-based amplification of circularized DNA"

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2480-1

    Error rate and mutation types of Droplet-CirSeq. a Error rate of Droplet-CirSeq and Cir-seq. The “1X allele” represents the bases that were supported at least by one circularized DNA, while the “2X allele” represents the bases that were supported by at least two different circularized DNAs. The error rate of Droplet-CirSeq was 5.23 X 10 -5 (±1.54 X 10 -5 ) at the “1X allele” criterion and 3.71 X 10 -6 (±2.34 X 10 -7 ) at the “2X allele” criterion. b Mutation types of Droplet-CirSeq. The error rates of most of the mutation types were lower than 3.00 X 10 -6 , but the error rates for the transitions C= > T and G= > A were almost one order of magnitude higher than the other types, and the other two transitions, A= > G and T= > C, also showed high error rates. The mutation pattern of the “2X allele” showed the same pattern as with the “1X allele”
    Figure Legend Snippet: Error rate and mutation types of Droplet-CirSeq. a Error rate of Droplet-CirSeq and Cir-seq. The “1X allele” represents the bases that were supported at least by one circularized DNA, while the “2X allele” represents the bases that were supported by at least two different circularized DNAs. The error rate of Droplet-CirSeq was 5.23 X 10 -5 (±1.54 X 10 -5 ) at the “1X allele” criterion and 3.71 X 10 -6 (±2.34 X 10 -7 ) at the “2X allele” criterion. b Mutation types of Droplet-CirSeq. The error rates of most of the mutation types were lower than 3.00 X 10 -6 , but the error rates for the transitions C= > T and G= > A were almost one order of magnitude higher than the other types, and the other two transitions, A= > G and T= > C, also showed high error rates. The mutation pattern of the “2X allele” showed the same pattern as with the “1X allele”

    Techniques Used: Mutagenesis

    Overview of Droplet-CirSeq. a Droplet-CirSeq workflow. Genomic DNA was sheared into fragments shorter than half the length of the sequencing read and then denatured into single-stranded DNA molecules that were circularized using single-strand DNA ligase. After eliminating the linear DNA using DNA exonucleases, the circularized single-stranded DNA was used for RCA (rolling circle replication). The circularized DNA and RCA reaction mix was added to a RainDrop Source chip to produce water-in-oil emulsion droplets. Generally, approximately 5 ~ 10 million droplets formed in approximately an hour in a 50 μl volume. The droplets containing RCA mix were allowed to continue to react for 4 ~ 16 h in order to amplify enough DNA for standard NGS library preparation in the following steps. Please note that the insert size of the standard NGS libraries must be larger than twice the length of the original circularized DNA to avoid sequencing the same DNA copy twice instead of sequencing two independent-amplified copies. b Error correction. Here is an example to explain the error correction strategy. Multiple copies of the original circularized DNA were examined in every PE read. “A” (red color) represents the base, which may have errors generated during PCR or sequencing. There will be three cases present in the sequencing result: AA (case 1), no errors; AB (case 2), one read error; and BB (case 3), two read errors. B stands for T/C /G. In the following bioinformatics analysis, Case 2 and Case 3 will be filtered except when BB has the same bases, such as TT, GG, or CC (false positive)
    Figure Legend Snippet: Overview of Droplet-CirSeq. a Droplet-CirSeq workflow. Genomic DNA was sheared into fragments shorter than half the length of the sequencing read and then denatured into single-stranded DNA molecules that were circularized using single-strand DNA ligase. After eliminating the linear DNA using DNA exonucleases, the circularized single-stranded DNA was used for RCA (rolling circle replication). The circularized DNA and RCA reaction mix was added to a RainDrop Source chip to produce water-in-oil emulsion droplets. Generally, approximately 5 ~ 10 million droplets formed in approximately an hour in a 50 μl volume. The droplets containing RCA mix were allowed to continue to react for 4 ~ 16 h in order to amplify enough DNA for standard NGS library preparation in the following steps. Please note that the insert size of the standard NGS libraries must be larger than twice the length of the original circularized DNA to avoid sequencing the same DNA copy twice instead of sequencing two independent-amplified copies. b Error correction. Here is an example to explain the error correction strategy. Multiple copies of the original circularized DNA were examined in every PE read. “A” (red color) represents the base, which may have errors generated during PCR or sequencing. There will be three cases present in the sequencing result: AA (case 1), no errors; AB (case 2), one read error; and BB (case 3), two read errors. B stands for T/C /G. In the following bioinformatics analysis, Case 2 and Case 3 will be filtered except when BB has the same bases, such as TT, GG, or CC (false positive)

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Next-Generation Sequencing, Amplification, Generated, Polymerase Chain Reaction

    a FN site distribution of the Droplet-CirSeq and Cir-seq. The SEQ_Bias sites are FN sites with a depth less than or equal to 30X. The STR_Bias sites are FN sites with a depth greater than 30X but were still not detected as SNPs due to DNA strand amplification bias. The 3 pg input Droplet-CirSeq method had a lower SEQ_Bias, indicating that it improved the amplification of the poorly amplified region. The 300 pg input Droplet-CirSeq method had a lower STR_Bias, indicating that greater input improved STR_Bias. b Mutation type of FP sites. c True positive SNP frequency distribution for Droplet-CirSeq and Cir-seq; the box width indicates the detected SNP number, the outliers were excluded. d Mutation frequency of FP sites. e FPR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern. f FNR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern
    Figure Legend Snippet: a FN site distribution of the Droplet-CirSeq and Cir-seq. The SEQ_Bias sites are FN sites with a depth less than or equal to 30X. The STR_Bias sites are FN sites with a depth greater than 30X but were still not detected as SNPs due to DNA strand amplification bias. The 3 pg input Droplet-CirSeq method had a lower SEQ_Bias, indicating that it improved the amplification of the poorly amplified region. The 300 pg input Droplet-CirSeq method had a lower STR_Bias, indicating that greater input improved STR_Bias. b Mutation type of FP sites. c True positive SNP frequency distribution for Droplet-CirSeq and Cir-seq; the box width indicates the detected SNP number, the outliers were excluded. d Mutation frequency of FP sites. e FPR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern. f FNR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern

    Techniques Used: Amplification, Mutagenesis

    14) Product Images from "Cloning and Variation of Ground State Intestinal Stem Cells"

    Article Title: Cloning and Variation of Ground State Intestinal Stem Cells

    Journal: Nature

    doi: 10.1038/nature14484

    ISC GS  tumorigenicity assays in immunodeficient mice a.  Quantification of tumor formation assesses at 4–16 weeks following subcutaneous innoculation of two million cells of the indicated ISC pedigrees at passage 6 or passage 25 at 4–16 weeks. “Pool” indicates total set of clones derived from P0 ileum culture prior to pedigree generation. “Cancer cells” refers to propagating cells from case of high-grade serous ovarian cancer.  b. Left , Histological section through site of injection of 1 million cells from pedigree 3.  Right,  Section of injection site stained with antibody (STEM121) to human epithelial cells (brown) revealing benign cysts. Scale bar, 15um.
    Figure Legend Snippet: ISC GS tumorigenicity assays in immunodeficient mice a. Quantification of tumor formation assesses at 4–16 weeks following subcutaneous innoculation of two million cells of the indicated ISC pedigrees at passage 6 or passage 25 at 4–16 weeks. “Pool” indicates total set of clones derived from P0 ileum culture prior to pedigree generation. “Cancer cells” refers to propagating cells from case of high-grade serous ovarian cancer. b. Left , Histological section through site of injection of 1 million cells from pedigree 3. Right, Section of injection site stained with antibody (STEM121) to human epithelial cells (brown) revealing benign cysts. Scale bar, 15um.

    Techniques Used: Mouse Assay, Clone Assay, Derivative Assay, Injection, Staining

    15) Product Images from "Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments"

    Article Title: Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments

    Journal: Environmental Microbiology

    doi: 10.1111/j.1462-2920.2010.02280.x

    Proposed role of 2-oxoglutarate (2OG) during cyanophage infection. A. In uninfected cyanobacteria, nitrogen limitation causes 2OG to accumulate, leading to 2OG-dependent binding of NtcA to promoters of nitrogen-stress genes, resulting in their expression. B. Phage infection draws down cellular nitrogen causing N-stress and likely leading to 2OG accumulation. Several cyanophage-encoded enzymes (in bold) suggest that increased 2OG may facilitate phage infection. First, a putative phytanoyl-CoA dioxygenase may convert 2OG to succinate, a major electron donor to respiratory electron transport in cyanobacteria ( Cooley and Vermaas, 2001 ) thus potentially generating energy for the infection process. Second, 2OG-dependent dioxygenase [2OG-Fe(II)] superfamily proteins may function in cyanophage DNA repair ( Weigele et al ., 2007 ). Third, cyanophage genomes have multiple NtcA promoters driving genes encoding diverse functions – possibly exploiting the host NtcA-driven N-stress response system.
    Figure Legend Snippet: Proposed role of 2-oxoglutarate (2OG) during cyanophage infection. A. In uninfected cyanobacteria, nitrogen limitation causes 2OG to accumulate, leading to 2OG-dependent binding of NtcA to promoters of nitrogen-stress genes, resulting in their expression. B. Phage infection draws down cellular nitrogen causing N-stress and likely leading to 2OG accumulation. Several cyanophage-encoded enzymes (in bold) suggest that increased 2OG may facilitate phage infection. First, a putative phytanoyl-CoA dioxygenase may convert 2OG to succinate, a major electron donor to respiratory electron transport in cyanobacteria ( Cooley and Vermaas, 2001 ) thus potentially generating energy for the infection process. Second, 2OG-dependent dioxygenase [2OG-Fe(II)] superfamily proteins may function in cyanophage DNA repair ( Weigele et al ., 2007 ). Third, cyanophage genomes have multiple NtcA promoters driving genes encoding diverse functions – possibly exploiting the host NtcA-driven N-stress response system.

    Techniques Used: Infection, Binding Assay, Expressing

    16) Product Images from "QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice"

    Article Title: QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.01634

    Relative expression of LOC_Os06g15370 (A) and LOC_Os06g15420 (B) after 48 h of trearment with 1 mM NH 4 NO 3 nutrient solution in GH998 and Y11. The X-axis represents different treatment stage; the Y-axis are scales of relative expression level. Error bars indicate standard deviations of independent biological replicates.
    Figure Legend Snippet: Relative expression of LOC_Os06g15370 (A) and LOC_Os06g15420 (B) after 48 h of trearment with 1 mM NH 4 NO 3 nutrient solution in GH998 and Y11. The X-axis represents different treatment stage; the Y-axis are scales of relative expression level. Error bars indicate standard deviations of independent biological replicates.

    Techniques Used: Expressing

    17) Product Images from "Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch"

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03868-8

    P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j ) are shown in Supplementary Fig. 8 . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P
    Figure Legend Snippet: P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j ) are shown in Supplementary Fig. 8 . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Techniques Used: Knock-In, Staining, Real-time Polymerase Chain Reaction

    18) Product Images from "Identification of genetic linkage group 1-linked sequences in Japanese eel (Anguilla japonica) by single chromosome sorting and sequencing"

    Article Title: Identification of genetic linkage group 1-linked sequences in Japanese eel (Anguilla japonica) by single chromosome sorting and sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197040

    Karyotype and FISH mapping probed with PCR amplicons of LG1-linked scaffolds in Japanese eel. Giemsa-stained karyotype of a wild-captured Japanese eel (A). Hybridization of rhodamine-labeled scaffold_127 amplicons (red signals) on a DAPI-stained metaphase spread (B). Hybridization of Alexa Fluor® 488-labeled scaffold_10233 (green signals) and rhodamine-labeled 12248 (red signals) amplicons on a DAPI-stained metaphase spread (C), and image of fluorescent signals only on the same metaphase spread (D). Arrowheads indicate hybridization signals. Scale bars represent 10 μm.
    Figure Legend Snippet: Karyotype and FISH mapping probed with PCR amplicons of LG1-linked scaffolds in Japanese eel. Giemsa-stained karyotype of a wild-captured Japanese eel (A). Hybridization of rhodamine-labeled scaffold_127 amplicons (red signals) on a DAPI-stained metaphase spread (B). Hybridization of Alexa Fluor® 488-labeled scaffold_10233 (green signals) and rhodamine-labeled 12248 (red signals) amplicons on a DAPI-stained metaphase spread (C), and image of fluorescent signals only on the same metaphase spread (D). Arrowheads indicate hybridization signals. Scale bars represent 10 μm.

    Techniques Used: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Staining, Hybridization, Labeling

    FISH mapping with putative LG1-linked scaffolds in Japanese eel. PCR amplicons from scaffold_12 (A), 135 and 297 (B), and 256 and 997 (C) are mapped to chromosome 5. The amplicons of scaffold_12 (A), 135 (B), and 256 (C) were labeled with rhodamine (red signals), and scaffold_297 (B) and 997 (C) were labeled with Alexa Fluor® 488 (green signals). Arrowheads indicate hybridization signals. Scale bars represent 10 μm.
    Figure Legend Snippet: FISH mapping with putative LG1-linked scaffolds in Japanese eel. PCR amplicons from scaffold_12 (A), 135 and 297 (B), and 256 and 997 (C) are mapped to chromosome 5. The amplicons of scaffold_12 (A), 135 (B), and 256 (C) were labeled with rhodamine (red signals), and scaffold_297 (B) and 997 (C) were labeled with Alexa Fluor® 488 (green signals). Arrowheads indicate hybridization signals. Scale bars represent 10 μm.

    Techniques Used: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Labeling, Hybridization

    19) Product Images from "An improvedPlasmodium cynomolgi genome assembly reveals an unexpected methyltransferase gene expansion"

    Article Title: An improvedPlasmodium cynomolgi genome assembly reveals an unexpected methyltransferase gene expansion

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.11864.1

    Expansion of Methyltransferase in P. cynomolgi and P. simiovale . ( A ) Tree of methyltransferase in Plasmodium, including the expansion of those genes in P. cynomolgi (36) and P. simiovale (at least 15). The closest core genes are PVP01_0943400 and PcyM_0947500. ( B ) Comparative view of P. cynomolgi and P. vivax on the locus of methyltransferase (*) of panel A. Interestingly, the locus in P. cynomolgi has an insertion with a subtelomeric gene that has a weak hit with to a putative DNA translocase Ftsk domain. Coverage plot mapped from P. cynomolgi reads (black), P. vivax (blue) and P. simiovale (magneta) is shown in log scale on P. cynomolgi . The methyltransferases are duplicated more than 35 times. As the height is roughly similar between the two duplications, we expect around the same number of methyltransferases in P. simiovale than in P. cynomolgi . The insert of the green gene is found just in P. cynomolgi, due to the missing coverage. The upper panel shows the distance of read pairs; the insertion of the region probably occurred after the duplication of the gene into the subtelomeres, as all reads from the duplications are connected over the insertion. The next core gene is also duplicated.
    Figure Legend Snippet: Expansion of Methyltransferase in P. cynomolgi and P. simiovale . ( A ) Tree of methyltransferase in Plasmodium, including the expansion of those genes in P. cynomolgi (36) and P. simiovale (at least 15). The closest core genes are PVP01_0943400 and PcyM_0947500. ( B ) Comparative view of P. cynomolgi and P. vivax on the locus of methyltransferase (*) of panel A. Interestingly, the locus in P. cynomolgi has an insertion with a subtelomeric gene that has a weak hit with to a putative DNA translocase Ftsk domain. Coverage plot mapped from P. cynomolgi reads (black), P. vivax (blue) and P. simiovale (magneta) is shown in log scale on P. cynomolgi . The methyltransferases are duplicated more than 35 times. As the height is roughly similar between the two duplications, we expect around the same number of methyltransferases in P. simiovale than in P. cynomolgi . The insert of the green gene is found just in P. cynomolgi, due to the missing coverage. The upper panel shows the distance of read pairs; the insertion of the region probably occurred after the duplication of the gene into the subtelomeres, as all reads from the duplications are connected over the insertion. The next core gene is also duplicated.

    Techniques Used:

    20) Product Images from "Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52"

    Article Title: Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12028

    Effects of loss of ID 4 on AR – FKBP 52 interaction and nuclear translocation in L+ns and L(−) ID 4 cells. (A) In vitro GST pull‐down assays were performed with purified, recombinant GST ‐tagged AR , His6‐ FKBP 52, and recombinant ID 4. Proteins were detected with respective primary antibodies to human AR , ID 4, and FKBP 52. (B) In vitro poly‐histidine pull‐down assays were performed with purified, recombinant His6‐tagged FKBP 52, recombinant full‐length ID 4, and truncated ID 4 constructs ID 4S73A ( ID 4 HLH mutant) and ID 4∆A mutant ( ID 4 in which the alanine tract was deleted) as indicated. Proteins were detected with respective primary antibodies. (C) Pull‐down assays using LNC aP and DU ‐145 whole‐cell lysates were performed with recombinant full‐length GST ‐ ID 4, or truncated GST ‐ ID 4 constructs ID 4S73A ( ID 4 HLH mutant) and ID 4∆A mutant. (D, E). The effects of loss of ID 4 on AR – FKBP 52 interaction and/or AR –Hsp90 complex dissociation were assessed in L+ns and L(−) ID 4 cells by co‐immunoprecipitation and western blot analysis. Lysates prepared from cells grown in 10% charcoal‐stripped fetal bovine serum (cs FBS ) in the absence or presence of R1881 (10 n m ) for 24 h were subjected to immunoprecipitation with either an antibody against FKBP 52 (D), or AR (E) and immunoblotted for the indicated proteins. (F) Cytoplasmic versus nuclear immunolocalization of AR and FKBP 52 in L+ns and L(−) ID 4 cells in response to R1881 (10 n m ). Topoisomerase (Topo1) and GAPDH were used to determine the purity and loading of nuclear and cytoplasmic extracts, respectively.
    Figure Legend Snippet: Effects of loss of ID 4 on AR – FKBP 52 interaction and nuclear translocation in L+ns and L(−) ID 4 cells. (A) In vitro GST pull‐down assays were performed with purified, recombinant GST ‐tagged AR , His6‐ FKBP 52, and recombinant ID 4. Proteins were detected with respective primary antibodies to human AR , ID 4, and FKBP 52. (B) In vitro poly‐histidine pull‐down assays were performed with purified, recombinant His6‐tagged FKBP 52, recombinant full‐length ID 4, and truncated ID 4 constructs ID 4S73A ( ID 4 HLH mutant) and ID 4∆A mutant ( ID 4 in which the alanine tract was deleted) as indicated. Proteins were detected with respective primary antibodies. (C) Pull‐down assays using LNC aP and DU ‐145 whole‐cell lysates were performed with recombinant full‐length GST ‐ ID 4, or truncated GST ‐ ID 4 constructs ID 4S73A ( ID 4 HLH mutant) and ID 4∆A mutant. (D, E). The effects of loss of ID 4 on AR – FKBP 52 interaction and/or AR –Hsp90 complex dissociation were assessed in L+ns and L(−) ID 4 cells by co‐immunoprecipitation and western blot analysis. Lysates prepared from cells grown in 10% charcoal‐stripped fetal bovine serum (cs FBS ) in the absence or presence of R1881 (10 n m ) for 24 h were subjected to immunoprecipitation with either an antibody against FKBP 52 (D), or AR (E) and immunoblotted for the indicated proteins. (F) Cytoplasmic versus nuclear immunolocalization of AR and FKBP 52 in L+ns and L(−) ID 4 cells in response to R1881 (10 n m ). Topoisomerase (Topo1) and GAPDH were used to determine the purity and loading of nuclear and cytoplasmic extracts, respectively.

    Techniques Used: Translocation Assay, In Vitro, Purification, Recombinant, Construct, Mutagenesis, Immunoprecipitation, Western Blot

    Inactivation of ID 4 in LNC aP cells promotes constitutive AR activation through FKBP 52. (A) PSA , TMPRSS 2, and AR gene expression levels in L+ns and L(−) ID 4 cells were assessed by q RT ‐ PCR analysis. Cells were treated for 24 h with increasing concentrations of MJC 13 as indicated above in the presence of 10% FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns and L(−) ID 4 (set to 1). (B) Immunoblot analysis of AR and AR ‐regulated proteins PSA and FKBP 51 in L+ns and L(−) ID 4 cells treated for 24 h with increasing concentrations of MJC 13 as indicated above. (C) Semiquantitative protein expression (from B) of PSA , FKBP 51, and AR protein levels was normalized to GAPDH and then to the individual protein levels in L+ns and L(−) ID 4 cells. (D, E) The AR transcriptional activity was determined by transiently transfecting L+ns and L(−) ID 4 cells with the AR response element‐driven luciferase reporter plasmid ( PSA luciferase), then treated with MJC 13 (30 μ m ) for 1 h followed by the addition of R1881 (1 n m ) or vehicle for additional 24 h. The data are normalized to Renilla luciferase. The mutated AR luciferase reporter plasmid ( ARR 3‐luciferase) was used as a negative control. The AR luciferase reporter activity in L+ns and L(−) ID 4 cells treated with MJC 13 was normalized to control L+ns and L(−) ID 4 cells. (F, G) Proliferation rate of L+ns and L(−) ID 4 cells treated with MJC 13 concentrations as indicated above in the absence or presence of R1881 (1 n m ) for 24 h. Data are presented as mean ± SEM ( n = 3; ***, *** ,a : P
    Figure Legend Snippet: Inactivation of ID 4 in LNC aP cells promotes constitutive AR activation through FKBP 52. (A) PSA , TMPRSS 2, and AR gene expression levels in L+ns and L(−) ID 4 cells were assessed by q RT ‐ PCR analysis. Cells were treated for 24 h with increasing concentrations of MJC 13 as indicated above in the presence of 10% FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns and L(−) ID 4 (set to 1). (B) Immunoblot analysis of AR and AR ‐regulated proteins PSA and FKBP 51 in L+ns and L(−) ID 4 cells treated for 24 h with increasing concentrations of MJC 13 as indicated above. (C) Semiquantitative protein expression (from B) of PSA , FKBP 51, and AR protein levels was normalized to GAPDH and then to the individual protein levels in L+ns and L(−) ID 4 cells. (D, E) The AR transcriptional activity was determined by transiently transfecting L+ns and L(−) ID 4 cells with the AR response element‐driven luciferase reporter plasmid ( PSA luciferase), then treated with MJC 13 (30 μ m ) for 1 h followed by the addition of R1881 (1 n m ) or vehicle for additional 24 h. The data are normalized to Renilla luciferase. The mutated AR luciferase reporter plasmid ( ARR 3‐luciferase) was used as a negative control. The AR luciferase reporter activity in L+ns and L(−) ID 4 cells treated with MJC 13 was normalized to control L+ns and L(−) ID 4 cells. (F, G) Proliferation rate of L+ns and L(−) ID 4 cells treated with MJC 13 concentrations as indicated above in the absence or presence of R1881 (1 n m ) for 24 h. Data are presented as mean ± SEM ( n = 3; ***, *** ,a : P

    Techniques Used: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Luciferase, Plasmid Preparation, Negative Control

    Hsp27 and FKBP 52 expression in L+ns and L(−) ID 4 cell lines. (A, B) Comparison of AR co‐chaperones Hsp27 (A) and FKBP 52 (B) mRNA levels in L+ns and L(−) ID 4 cells by q RT ‐ PCR analysis. Cells were treated for 24 h in the absence or presence of R1881 (10 n m ) in 10% cs FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns (set to 1). (C) Protein levels of AR co‐chaperones Hsp27, phosphorylated (p) Hsp27 [Ser82], and FKBP 52 in L+ns and L(−) ID 4 cells. (D) Semiquantitative FKBP 52, Hsp27, and P‐Hsp27 (S‐82) protein levels normalized to GAPDH and then to the individual protein levels in L+ns and L(−) ID 4 cells. Data are mean ± SEM ( n = 3; ***, *** ,a : P
    Figure Legend Snippet: Hsp27 and FKBP 52 expression in L+ns and L(−) ID 4 cell lines. (A, B) Comparison of AR co‐chaperones Hsp27 (A) and FKBP 52 (B) mRNA levels in L+ns and L(−) ID 4 cells by q RT ‐ PCR analysis. Cells were treated for 24 h in the absence or presence of R1881 (10 n m ) in 10% cs FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns (set to 1). (C) Protein levels of AR co‐chaperones Hsp27, phosphorylated (p) Hsp27 [Ser82], and FKBP 52 in L+ns and L(−) ID 4 cells. (D) Semiquantitative FKBP 52, Hsp27, and P‐Hsp27 (S‐82) protein levels normalized to GAPDH and then to the individual protein levels in L+ns and L(−) ID 4 cells. Data are mean ± SEM ( n = 3; ***, *** ,a : P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Expression of AR and AR ‐regulated genes/proteins in L+ns and L(−) ID 4 cell lines. (A) AR mRNA levels in L+ns and L(−) ID 4 cells were determined by quantitative real‐time (q RT )‐ PCR analysis in response to R1881 (R, 10 n m ). Data ( n = 3) are normalized to GAPDH followed by relative expression compared with the AR gene in L+ns (set to 1). (B) Immunoblot analysis of AR in response to cycloheximide ( CHX , 100 μg·mL −1 ) treatment. Cells were treated with cycloheximide for the indicated time points (0, 6, 12, 24, and 30 h) followed by AR immunoblot analysis. (C) Semiquantitative AR protein levels (from B) normalized to GAPDH (loading control) and then to the individual AR protein levels in L+ns and L(−) ID 4 cells. (D–F) q RT ‐ PCR analysis of AR ‐regulated genes PSA , FKBP 51, and ARD 1 in L+ns and L(−) ID 4 cells in response to R1881 (10 n m ) in 10% cs FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns (set to 1). (G) AR ‐dependent protein expression of PSA , FKBP 51, and ARD 1 in L+ns and L(−) ID 4 cells cultured for 24 h in 10% cs FBS before treatment with R1881 (10 n m ) and/or R1881 (10 n m ) ± Casodex (30 μ m , antiandrogen) for another 24 h. (H) Semiquantitative FKBP 51, ARD 1, and PSA protein levels normalized to GAPDH (loading control) and then to the individual protein levels in L+ns and L(−) ID 4 cells. Data are mean ± SEM ( n = 3; *: P
    Figure Legend Snippet: Expression of AR and AR ‐regulated genes/proteins in L+ns and L(−) ID 4 cell lines. (A) AR mRNA levels in L+ns and L(−) ID 4 cells were determined by quantitative real‐time (q RT )‐ PCR analysis in response to R1881 (R, 10 n m ). Data ( n = 3) are normalized to GAPDH followed by relative expression compared with the AR gene in L+ns (set to 1). (B) Immunoblot analysis of AR in response to cycloheximide ( CHX , 100 μg·mL −1 ) treatment. Cells were treated with cycloheximide for the indicated time points (0, 6, 12, 24, and 30 h) followed by AR immunoblot analysis. (C) Semiquantitative AR protein levels (from B) normalized to GAPDH (loading control) and then to the individual AR protein levels in L+ns and L(−) ID 4 cells. (D–F) q RT ‐ PCR analysis of AR ‐regulated genes PSA , FKBP 51, and ARD 1 in L+ns and L(−) ID 4 cells in response to R1881 (10 n m ) in 10% cs FBS . Data were normalized to GAPDH followed by relative expression compared with the respective genes in L+ns (set to 1). (G) AR ‐dependent protein expression of PSA , FKBP 51, and ARD 1 in L+ns and L(−) ID 4 cells cultured for 24 h in 10% cs FBS before treatment with R1881 (10 n m ) and/or R1881 (10 n m ) ± Casodex (30 μ m , antiandrogen) for another 24 h. (H) Semiquantitative FKBP 51, ARD 1, and PSA protein levels normalized to GAPDH (loading control) and then to the individual protein levels in L+ns and L(−) ID 4 cells. Data are mean ± SEM ( n = 3; *: P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    21) Product Images from "Performance of four modern whole genome amplification methods for copy number variant detection in single cells"

    Article Title: Performance of four modern whole genome amplification methods for copy number variant detection in single cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03711-y

    Experimental design. Samples consisting of 1, 3 or 5 cells were collected from the Loucy cell line using micromanipulation for each WGA method in triplicate. Cells were amplified with either Ampli-1, REPLI-g or DOPlify, followed by PCR-free Illumina library preparation and sequencing. A fourth method, Picoseq, performs WGA and library preparation simultaneously, without the need for a separate library preparation. A bulk DNA sample was extracted from 5 * 10 6 Loucy cells using a column-based extraction method from Qiagen, also followed by PCR-free Illumina library preparation and sequencing.
    Figure Legend Snippet: Experimental design. Samples consisting of 1, 3 or 5 cells were collected from the Loucy cell line using micromanipulation for each WGA method in triplicate. Cells were amplified with either Ampli-1, REPLI-g or DOPlify, followed by PCR-free Illumina library preparation and sequencing. A fourth method, Picoseq, performs WGA and library preparation simultaneously, without the need for a separate library preparation. A bulk DNA sample was extracted from 5 * 10 6 Loucy cells using a column-based extraction method from Qiagen, also followed by PCR-free Illumina library preparation and sequencing.

    Techniques Used: Micromanipulation, Whole Genome Amplification, Amplification, Polymerase Chain Reaction, Sequencing

    22) Product Images from "Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice"

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky456

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Figure Legend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Techniques Used: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    Heart Sod2 knockout mice display severe dilated cardiomyopathy. ( A ) Western blot analysis of SOD2 protein levels from purified mitochondria of control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre) (9–11 week old). ATP5A was used as a loading control. ( B ) Vertical sections through the midpoint of paraffin embedded hearts stained with hematoxylin and eosin staining. Control (pp, 11-week old) and Sod2 loxP × Ckmm cre (pp, cre, 10-week old). Scale bar represents 1 mm. ( C ) Heart weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 22, male n = 35, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 2, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 28, male n = 15, 9–10 week old). D. Body weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 26, male n = 36, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 3, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 30, male n = 19, 9–10 week old). Whiskers represent min and max values, horizontal lines medians; **** P
    Figure Legend Snippet: Heart Sod2 knockout mice display severe dilated cardiomyopathy. ( A ) Western blot analysis of SOD2 protein levels from purified mitochondria of control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre) (9–11 week old). ATP5A was used as a loading control. ( B ) Vertical sections through the midpoint of paraffin embedded hearts stained with hematoxylin and eosin staining. Control (pp, 11-week old) and Sod2 loxP × Ckmm cre (pp, cre, 10-week old). Scale bar represents 1 mm. ( C ) Heart weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 22, male n = 35, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 2, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 28, male n = 15, 9–10 week old). D. Body weight of control (pp), heterozygous Sod2 loxP x Ckmm cre and homozygous Sod2 loxP x Ckmm cre (pp, cre) mice. White box indicates control mice (pp female n = 26, male n = 36, 9–11 week old), light gray box indicates heterozygous Sod2 loxP x Ckmm cre mice (male +p, cre, n = 3, 9-week old) and dark gray box indicates homozygous Sod2 loxP x Ckmm cre mice (pp, cre, female n = 30, male n = 19, 9–10 week old). Whiskers represent min and max values, horizontal lines medians; **** P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot, Purification, Staining

    POLγ steady-state levels are decreased in heart Sod2 knockout mice while POLRMT levels are increased. Western blot analysis of proteins involved in replication and transcription from purified heart mitochondria from control (pp) and Sod2 loxP x Ckmm cre (pp, cre) mice (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls.
    Figure Legend Snippet: POLγ steady-state levels are decreased in heart Sod2 knockout mice while POLRMT levels are increased. Western blot analysis of proteins involved in replication and transcription from purified heart mitochondria from control (pp) and Sod2 loxP x Ckmm cre (pp, cre) mice (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls.

    Techniques Used: Knock-Out, Mouse Assay, Western Blot, Purification, Staining

    [4Fe–4S] cluster proteins are severely affected in heart Sod2 knockout mice indicating strong increase in superoxide levels. ( A ) Aconitase activity from purified mitochondria from control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre). White bar indicates activity in control samples ( n = 6, 9–10 week old) and gray bar in Sod2 loxP x Ckmm cre samples ( n = 6, 9–12 week old). Activity is normalized to control. ( B ) Western blot analysis of ACO 2 (aconitase) protein levels from purified mitochondria of control (pp) and Sod2 loxP x Ckmm cre mice (pp, cre) (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls. ( C ) Oxygen consumption rate of isolated heart mitochondria from control (pp, white bars, n = 9, 9–11 week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars, n = 9, 9–12 week old). Isolated mitochondria were incubated with complex I (PMG) or complex II (SUCC) substrates. Each set of substrates was successively combined with ADP (to assess the phosphorylating respiration, PMG3, SUCC3), oligomycin (to assess the non-phosphorylating respiration PMG4, SUCC4) and CCCP (to assess uncoupled respiration PMGc, SUCCc). ( D ) Activity of the respiratory chain complexes I (CI), II (CII), IV (CIV) and the activity from complex II to III (CII-III) of heart mitochondria from control (pp, write bars, n = 3, 11-week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars n = 3, 11–12 week old). Citrate synthase activity (CS) was used as a control. Error bars represent SD. * P
    Figure Legend Snippet: [4Fe–4S] cluster proteins are severely affected in heart Sod2 knockout mice indicating strong increase in superoxide levels. ( A ) Aconitase activity from purified mitochondria from control (pp) and Sod2 loxP × Ckmm cre mice (pp, cre). White bar indicates activity in control samples ( n = 6, 9–10 week old) and gray bar in Sod2 loxP x Ckmm cre samples ( n = 6, 9–12 week old). Activity is normalized to control. ( B ) Western blot analysis of ACO 2 (aconitase) protein levels from purified mitochondria of control (pp) and Sod2 loxP x Ckmm cre mice (pp, cre) (9–10 week old). ATP5A and Coomassie-stained membrane were used as loading controls. ( C ) Oxygen consumption rate of isolated heart mitochondria from control (pp, white bars, n = 9, 9–11 week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars, n = 9, 9–12 week old). Isolated mitochondria were incubated with complex I (PMG) or complex II (SUCC) substrates. Each set of substrates was successively combined with ADP (to assess the phosphorylating respiration, PMG3, SUCC3), oligomycin (to assess the non-phosphorylating respiration PMG4, SUCC4) and CCCP (to assess uncoupled respiration PMGc, SUCCc). ( D ) Activity of the respiratory chain complexes I (CI), II (CII), IV (CIV) and the activity from complex II to III (CII-III) of heart mitochondria from control (pp, write bars, n = 3, 11-week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars n = 3, 11–12 week old). Citrate synthase activity (CS) was used as a control. Error bars represent SD. * P

    Techniques Used: Knock-Out, Mouse Assay, Activity Assay, Purification, Western Blot, Staining, Isolation, Incubation

    Mitochodrial BER deficient mice do not accumulate point mutations to mtDNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Data is quality filtered and minimum variant allele frequency is set to 0.5%. For the unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (pp n = 4 or ++ n = 3, 8–12 week old), light gray circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4 or +p dd n = 2 or +p cre+ dd n = 1, 8–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 7, 9–10 week old). Horizontal lines represent means, one-way ANOVA, Tukey's multiple comparison test. ( B ) Mutation profile of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Samples as in A. Horizontal lines represent mean. For only quality-filtered data see Supplementary Figure S4 .
    Figure Legend Snippet: Mitochodrial BER deficient mice do not accumulate point mutations to mtDNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Data is quality filtered and minimum variant allele frequency is set to 0.5%. For the unique mutation load each mutation is counted only once, reflecting how many times a specific mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. White circles indicate samples from controls (pp n = 4 or ++ n = 3, 8–12 week old), light gray circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4 or +p dd n = 2 or +p cre+ dd n = 1, 8–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 7, 9–10 week old). Horizontal lines represent means, one-way ANOVA, Tukey's multiple comparison test. ( B ) Mutation profile of mtDNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS with Illumina sequencing. The sequencing was carried out from purified mtDNA from heart. Samples as in A. Horizontal lines represent mean. For only quality-filtered data see Supplementary Figure S4 .

    Techniques Used: Mouse Assay, Mutagenesis, Sequencing, Purification, Variant Assay

    Mitochondrial BER deficient mice do not accumulate point mutations of mtRNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. For the unique mutation load each specific mutation is counted only once, reflecting how many times a mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre mice from heart. White circles indicate samples from controls (+p n = 1 pp n = 2, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre n = 3, 10–11 week old). ( B ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. White circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 4, 9–10 week old). * P
    Figure Legend Snippet: Mitochondrial BER deficient mice do not accumulate point mutations of mtRNA even in the presence of increased oxidative stress. ( A ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. For the unique mutation load each specific mutation is counted only once, reflecting how many times a mutation has occurred. For the total mutation load each mutation is counted as many times as it is seen, reflecting the clonal expansion of mutations. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre mice from heart. White circles indicate samples from controls (+p n = 1 pp n = 2, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre n = 3, 10–11 week old). ( B ) Mutation load of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. Illumina sequencing was carried out from total RNA considering only the reads that map to mtDNA for variant calling. Data is quality filtered. Mutation profile of mtRNA from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice from heart. White circles indicate samples from Sod2 loxP x Ogg1 dMTS mice (pp dd n = 4, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre x Ogg1 dMTS mice (pp, cre dd, n = 4, 9–10 week old). * P

    Techniques Used: Mouse Assay, Mutagenesis, Sequencing, Variant Assay

    Heart Sod2 knockout mice show global decrease in complex I proteins and indications of general mitochondrial stress in label-free quantitative proteomics. Heat map of selected proteins from label-free quantitative proteomic analysis of Percoll gradient purified heart mitochondria from controls (pp, 8–9 week old) and Sod2 loxP × Ckmm cre mice (pp, cre, 9–10 week old). Changes in the protein steady-state levels are blotted as Z -scores. Blue indicates decreased and red increased steady-state level from the global mean across all samples. Abundances of all the presented proteins were significantly changed, with Benjamini–Hochberg adjusted P -values of
    Figure Legend Snippet: Heart Sod2 knockout mice show global decrease in complex I proteins and indications of general mitochondrial stress in label-free quantitative proteomics. Heat map of selected proteins from label-free quantitative proteomic analysis of Percoll gradient purified heart mitochondria from controls (pp, 8–9 week old) and Sod2 loxP × Ckmm cre mice (pp, cre, 9–10 week old). Changes in the protein steady-state levels are blotted as Z -scores. Blue indicates decreased and red increased steady-state level from the global mean across all samples. Abundances of all the presented proteins were significantly changed, with Benjamini–Hochberg adjusted P -values of

    Techniques Used: Knock-Out, Mouse Assay, Purification

    De novo replication is affected in heart Sod2 knockout mice. ( A ) Representative experiment of in organello replication assay in heart Sod2 loxP x Ckmm cre mice. The mtDNA was radioactively labeled in isolated mitochondria, purified and half of it was boiled to release newly synthesized 7S DNA. Samples were separated on an agarose gel and transferred to a membrane. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. ( B ) Quantification of de novo replication, the relative incorporation of radioactivity into mtDNA and newly synthesized 7S DNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of mtDNA that was probed from the same membrane after the de novo signal could not be detected anymore. White circles indicate samples from controls (pp, n = 9, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 9, 9–10 week old). ( C ) Representative experiment of in organello transcription assay from Sod2 loxP x Ckmm cre mice. The mtRNA was radioactively labeled in isolated mitochondria and half of the sample was purified (pulse). Another half was incubated with non-radioactive UTP for 2 h (chase) and purified, and then samples were analyzed with northern blotting. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. ( D ) Quantification of de novo transcription, the relative incorporation of radioactivity into mtRNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of CytB that was probed from the same membrane after the de novo signal could not be detected anymore. See Supplementary Figure S7 , which shows that CytB levels do not change in Sod2 loxP x Ckmm cre mice. White circles indicate samples from controls (pp, n = 12, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 12, 9–10 week old). Horizontal lines represent means, error bars represent SD, **** P
    Figure Legend Snippet: De novo replication is affected in heart Sod2 knockout mice. ( A ) Representative experiment of in organello replication assay in heart Sod2 loxP x Ckmm cre mice. The mtDNA was radioactively labeled in isolated mitochondria, purified and half of it was boiled to release newly synthesized 7S DNA. Samples were separated on an agarose gel and transferred to a membrane. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. ( B ) Quantification of de novo replication, the relative incorporation of radioactivity into mtDNA and newly synthesized 7S DNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of mtDNA that was probed from the same membrane after the de novo signal could not be detected anymore. White circles indicate samples from controls (pp, n = 9, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 9, 9–10 week old). ( C ) Representative experiment of in organello transcription assay from Sod2 loxP x Ckmm cre mice. The mtRNA was radioactively labeled in isolated mitochondria and half of the sample was purified (pulse). Another half was incubated with non-radioactive UTP for 2 h (chase) and purified, and then samples were analyzed with northern blotting. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. ( D ) Quantification of de novo transcription, the relative incorporation of radioactivity into mtRNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of CytB that was probed from the same membrane after the de novo signal could not be detected anymore. See Supplementary Figure S7 , which shows that CytB levels do not change in Sod2 loxP x Ckmm cre mice. White circles indicate samples from controls (pp, n = 12, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 12, 9–10 week old). Horizontal lines represent means, error bars represent SD, **** P

    Techniques Used: Knock-Out, Mouse Assay, Labeling, Isolation, Purification, Synthesized, Agarose Gel Electrophoresis, Staining, Radioactivity, Incubation, Northern Blot

    23) Product Images from "Diagnostic value and lymph node metastasis prediction of a custom-made panel (thyroline) in thyroid cancer"

    Article Title: Diagnostic value and lymph node metastasis prediction of a custom-made panel (thyroline) in thyroid cancer

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6493

    Gene mutations and fusions in subtypes of TC and workflow of NGS. (A) BRAF, RAS, TERT, ETV6, EIF1AX, GNAS, PIK3CA, TP53 and NTRK1 mutations, as well as RET and ALK fusions, were found in PTC. BRAF, TERT, ALK fusion, GNAS, AKT1, PIK3CA, TP53 and PTEN were found in ATC. RAS, TERT, TSHR, GNAS, PENT and TP53 were found in FTC, while only RET and RAS mutations were found in MTC. (B) FFPE samples were obtained from 98 thyroid nodule patients, which was followed by CTC enumeration on NanoVelcro Chips. After collecting clinical information, we analyzed the correlation between pathological information and NGS results. (C) DNA from FFPE tissue was amplified for enrichment of target regions in a multiplex PCR reaction. Then, the library was prepared by ligating the PCR amplicons into platform-specific adapters and adding bar codes for specimen multiplexing. Finally, the library was enriched by clonal amplification (emPCR) and sequenced by massively parallel sequencing on the Ion Torrent PGM. The data analysis and variant calling were performed using bioinformatic pipelines followed by a custom SeqReporter algorithm for filtering and annotation of genetic variants. TC, thyroid cancer; NGS, next-generation sequencing; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; FFPE, formalin-fixed, paraffin-embedded.
    Figure Legend Snippet: Gene mutations and fusions in subtypes of TC and workflow of NGS. (A) BRAF, RAS, TERT, ETV6, EIF1AX, GNAS, PIK3CA, TP53 and NTRK1 mutations, as well as RET and ALK fusions, were found in PTC. BRAF, TERT, ALK fusion, GNAS, AKT1, PIK3CA, TP53 and PTEN were found in ATC. RAS, TERT, TSHR, GNAS, PENT and TP53 were found in FTC, while only RET and RAS mutations were found in MTC. (B) FFPE samples were obtained from 98 thyroid nodule patients, which was followed by CTC enumeration on NanoVelcro Chips. After collecting clinical information, we analyzed the correlation between pathological information and NGS results. (C) DNA from FFPE tissue was amplified for enrichment of target regions in a multiplex PCR reaction. Then, the library was prepared by ligating the PCR amplicons into platform-specific adapters and adding bar codes for specimen multiplexing. Finally, the library was enriched by clonal amplification (emPCR) and sequenced by massively parallel sequencing on the Ion Torrent PGM. The data analysis and variant calling were performed using bioinformatic pipelines followed by a custom SeqReporter algorithm for filtering and annotation of genetic variants. TC, thyroid cancer; NGS, next-generation sequencing; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; FFPE, formalin-fixed, paraffin-embedded.

    Techniques Used: Next-Generation Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Multiplex Assay, Polymerase Chain Reaction, Multiplexing, Sequencing, Variant Assay

    24) Product Images from "Circular DNA elements of chromosomal origin are common in healthy human somatic tissue"

    Article Title: Circular DNA elements of chromosomal origin are common in healthy human somatic tissue

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03369-8

    Circle-Seq method for mapping of eccDNA. a Leukocyte and muscle samples from 16 healthy subjects (T1–T16, n = 16). b Purification of eccDNA through column separation, exonuclease treatment, and rolling-circle amplification. c Detection of eccDNA based on structural-read variants and coverage (soft-clipped, split, red; concordant, gray; and discordant reads, blue). d Read coverage display (log-scale) at the titin gene, TTN , from muscle samples. e EccDNA from T5 and T6 illustrated (black boxes, exons), outward PCR validation (blue arrows), inward PCR (black arrows), and gel-image of T5 and T6 PCR products next to controls: GD, human genomic DNA; φ , phi29-amplified eccDNA sample without detected [ TTN circle ]. f Sequence of [ TTN circle exon 44-52 ] T6 PCR product at junction
    Figure Legend Snippet: Circle-Seq method for mapping of eccDNA. a Leukocyte and muscle samples from 16 healthy subjects (T1–T16, n = 16). b Purification of eccDNA through column separation, exonuclease treatment, and rolling-circle amplification. c Detection of eccDNA based on structural-read variants and coverage (soft-clipped, split, red; concordant, gray; and discordant reads, blue). d Read coverage display (log-scale) at the titin gene, TTN , from muscle samples. e EccDNA from T5 and T6 illustrated (black boxes, exons), outward PCR validation (blue arrows), inward PCR (black arrows), and gel-image of T5 and T6 PCR products next to controls: GD, human genomic DNA; φ , phi29-amplified eccDNA sample without detected [ TTN circle ]. f Sequence of [ TTN circle exon 44-52 ] T6 PCR product at junction

    Techniques Used: Purification, Amplification, Polymerase Chain Reaction, Sequencing

    EccDNA validation and DNA deletion. a Gel images for a validated subset ( n = 9) of eccDNAs by outward PCR (blue arrows), inward PCR (black arrows), gel electrophoresis, and Sanger sequencing. EccDNAs are named according to gene content; black boxes, exons. Template: T1–T16, muscle B1–B16 leukocyte, phi29 (φ) amplified eccDNA, GD genomic DNA, NTC nontemplate control. Sequenced PCR products are in boxes and alignment of resultant sequence are red and blue lines. b Confirmed verification of DAZ4 ∆ exon 18 deletion and c [ DAZ4 circle exon 18 ] at identical coordinates within the DAZ4 locus by inward and outward PCR (oligos arrows, inward red, outward, blue)
    Figure Legend Snippet: EccDNA validation and DNA deletion. a Gel images for a validated subset ( n = 9) of eccDNAs by outward PCR (blue arrows), inward PCR (black arrows), gel electrophoresis, and Sanger sequencing. EccDNAs are named according to gene content; black boxes, exons. Template: T1–T16, muscle B1–B16 leukocyte, phi29 (φ) amplified eccDNA, GD genomic DNA, NTC nontemplate control. Sequenced PCR products are in boxes and alignment of resultant sequence are red and blue lines. b Confirmed verification of DAZ4 ∆ exon 18 deletion and c [ DAZ4 circle exon 18 ] at identical coordinates within the DAZ4 locus by inward and outward PCR (oligos arrows, inward red, outward, blue)

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Amplification

    25) Product Images from "Dual origin of relapses in retinoic-acid resistant acute promyelocytic leukemia"

    Article Title: Dual origin of relapses in retinoic-acid resistant acute promyelocytic leukemia

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04384-5

    Graphic summary of the exome analysis of relapsing APLs. a Number and type of somatic alterations identified at diagnosis (upper part) and acquired at relapse (lower part) for each patient. ND* indicates sample pairs with no available remission germline DNA, precluding determination of diagnostic alterations. b Somatic mutations (left) and copy-number alterations (right) observed at diagnosis (upper part) or relapse (lower part) at least twice in the study. Note the unexpected high prevalence and molecular variety of WT1 alterations
    Figure Legend Snippet: Graphic summary of the exome analysis of relapsing APLs. a Number and type of somatic alterations identified at diagnosis (upper part) and acquired at relapse (lower part) for each patient. ND* indicates sample pairs with no available remission germline DNA, precluding determination of diagnostic alterations. b Somatic mutations (left) and copy-number alterations (right) observed at diagnosis (upper part) or relapse (lower part) at least twice in the study. Note the unexpected high prevalence and molecular variety of WT1 alterations

    Techniques Used: Diagnostic Assay

    26) Product Images from "Sequencing of intraductal biopsies is feasible and potentially impacts clinical management of patients with indeterminate biliary stricture and cholangiocarcinoma"

    Article Title: Sequencing of intraductal biopsies is feasible and potentially impacts clinical management of patients with indeterminate biliary stricture and cholangiocarcinoma

    Journal: Clinical and Translational Gastroenterology

    doi: 10.1038/s41424-018-0015-6

    From endoscopy to targeted sequencing of intraductal biopsies. Representative results of endoscopical, histopathological and molecular analysis of Patient 1. In fluoroscopy ( a ), a high-grade stenosis of the distal common bile duct with concomitant intra- and extrahepatic cholestasis was diagnosed. In cholangioscopy ( b ), an ulcerative lesion highly suspicious for malignancy was identified to cause the biliary stricture. Histopathological analysis of hematoxylin–eosin stained sections of the biopsy ( c 50×, d 200×) was inconclusive: a definite differentiation of reactive and dysplastic alterations of the biliary epithelium was not possible due to artificial degradation of the obtained tissue. Due to clinical suspicion for malignancy, surgery was performed and histopathological analysis established diagnosis of adenocarcinoma ( e 50×, f 200×). After targeted sequencing ( g ), the same three mutations were observed in both the intraductal biopsy and the tumor sample such as shown for the mutation in SMAD4 (Chr.18:48604788, A > C), which was observed in both sequencing libraries of the tumor (Tu-a, Tu-b) as well as the biopsy sample (Bx-a, Bx-b). There was no mutation observed in any library of the non-tumor control sample (NT-a, NT-b)
    Figure Legend Snippet: From endoscopy to targeted sequencing of intraductal biopsies. Representative results of endoscopical, histopathological and molecular analysis of Patient 1. In fluoroscopy ( a ), a high-grade stenosis of the distal common bile duct with concomitant intra- and extrahepatic cholestasis was diagnosed. In cholangioscopy ( b ), an ulcerative lesion highly suspicious for malignancy was identified to cause the biliary stricture. Histopathological analysis of hematoxylin–eosin stained sections of the biopsy ( c 50×, d 200×) was inconclusive: a definite differentiation of reactive and dysplastic alterations of the biliary epithelium was not possible due to artificial degradation of the obtained tissue. Due to clinical suspicion for malignancy, surgery was performed and histopathological analysis established diagnosis of adenocarcinoma ( e 50×, f 200×). After targeted sequencing ( g ), the same three mutations were observed in both the intraductal biopsy and the tumor sample such as shown for the mutation in SMAD4 (Chr.18:48604788, A > C), which was observed in both sequencing libraries of the tumor (Tu-a, Tu-b) as well as the biopsy sample (Bx-a, Bx-b). There was no mutation observed in any library of the non-tumor control sample (NT-a, NT-b)

    Techniques Used: Sequencing, Staining, Mutagenesis

    27) Product Images from "Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA"

    Article Title: Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1334024

    Relationship between plasma cfDNA input and post-BSC DNA recovery. (A) Post-BSC cfDNA recovery vs. pre-BSC cfDNA input quantity (measured using ”Chr3-assay” and “MYOD1-assay”) for 6 BSC kits. (B) BSC kit efficiency displayed as percentage of fully un-converted cfDNA in the total converted cfDNA after BSC.
    Figure Legend Snippet: Relationship between plasma cfDNA input and post-BSC DNA recovery. (A) Post-BSC cfDNA recovery vs. pre-BSC cfDNA input quantity (measured using ”Chr3-assay” and “MYOD1-assay”) for 6 BSC kits. (B) BSC kit efficiency displayed as percentage of fully un-converted cfDNA in the total converted cfDNA after BSC.

    Techniques Used:

    28) Product Images from "Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences"

    Article Title: Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172769

    Base constitution surrounding the 3’ end of the sequenced fragments in SMART based libraries. (A) Sequence logo representation of the information content of the region surrounding the end of the second read for the forward (left) and reverse (right) strands in a HCT116 sample prepared with the SMART based library protocol ( S1 Table ). Similar results were obtained with all other SMART based samples. Each sequence logo is based on 1,000 randomly chosen reads. (B) IGV representation of two typical genomic regions (taken from the same SMART based library as in A), in which multiple reads ended at the same position. The red and blue rectangles represent the locations of the second reads in each pair for the forward (left) and the reverse (right) strands respectively. The small bars below the reads represent individual bases which are color coded. Note that immediately after the reads there are tracts of poly dT and poly dA for the forward and reverse strands, respectively. The sequence logos below the IGV tracts represent the information content as in A, for 1,000 randomly chosen genomic regions out of ~300,000 in which at least 5 reads were mapped.
    Figure Legend Snippet: Base constitution surrounding the 3’ end of the sequenced fragments in SMART based libraries. (A) Sequence logo representation of the information content of the region surrounding the end of the second read for the forward (left) and reverse (right) strands in a HCT116 sample prepared with the SMART based library protocol ( S1 Table ). Similar results were obtained with all other SMART based samples. Each sequence logo is based on 1,000 randomly chosen reads. (B) IGV representation of two typical genomic regions (taken from the same SMART based library as in A), in which multiple reads ended at the same position. The red and blue rectangles represent the locations of the second reads in each pair for the forward (left) and the reverse (right) strands respectively. The small bars below the reads represent individual bases which are color coded. Note that immediately after the reads there are tracts of poly dT and poly dA for the forward and reverse strands, respectively. The sequence logos below the IGV tracts represent the information content as in A, for 1,000 randomly chosen genomic regions out of ~300,000 in which at least 5 reads were mapped.

    Techniques Used: Sequencing

    Bias toward Poly dT tracts in SMART-DNA libraries. The number of reads (per million reads) mapped adjacent to poly dN tracts (≥12) for the forward and reverse strands is reported. These numbers are further normalized for the genomic frequencies of such tracts (see methods ). Data is shown for the second read in each pair (A-B), for the first read (D,E,G) and for random locations (shift of 1000 bps, see methods ) (C,F). The analysis was done for SMART-DNA based library preparation (A,D), for ligation based method (B,E) and for SMART-RNA library preparation (G). The distance between the read to the poly dN tract was set to be either 1 nucleotide (A-C) or 250 nucleotides (D-G). The data presented is for HCT116 genomic DNA sequenced by us (A, D), obtained from the Aladgem lab (B,E) and for RNA seq libraries (G) downloaded from [ 17 ]. Note that the SMART-RNA library was prepared by annealing a primer to poly dA tracts rather than to poly dT tracts as with other SMART based libraries. We tested the statistical significance of the deviation from a distribution that is based on the frequency of occurrences in the genome, using the Chi squared goodness of fit test. The effect sizes (ϕ) (see methods ) are shown in each graph. The data presented is for a single library from each type. Similar results were obtained for additional libraries ( S3 Fig ).
    Figure Legend Snippet: Bias toward Poly dT tracts in SMART-DNA libraries. The number of reads (per million reads) mapped adjacent to poly dN tracts (≥12) for the forward and reverse strands is reported. These numbers are further normalized for the genomic frequencies of such tracts (see methods ). Data is shown for the second read in each pair (A-B), for the first read (D,E,G) and for random locations (shift of 1000 bps, see methods ) (C,F). The analysis was done for SMART-DNA based library preparation (A,D), for ligation based method (B,E) and for SMART-RNA library preparation (G). The distance between the read to the poly dN tract was set to be either 1 nucleotide (A-C) or 250 nucleotides (D-G). The data presented is for HCT116 genomic DNA sequenced by us (A, D), obtained from the Aladgem lab (B,E) and for RNA seq libraries (G) downloaded from [ 17 ]. Note that the SMART-RNA library was prepared by annealing a primer to poly dA tracts rather than to poly dT tracts as with other SMART based libraries. We tested the statistical significance of the deviation from a distribution that is based on the frequency of occurrences in the genome, using the Chi squared goodness of fit test. The effect sizes (ϕ) (see methods ) are shown in each graph. The data presented is for a single library from each type. Similar results were obtained for additional libraries ( S3 Fig ).

    Techniques Used: Ligation, RNA Sequencing Assay

    29) Product Images from "Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch"

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03868-8

    JMJD1A demethylates H3K9me2 on beige-selective genes. a RNA-seq heat map depicting expression ratio comparison between beige and white adipocytes, differentiated from im-scWATs under rosiglitazone (ROS) plus or minus condition (left). RNA-seq heat map of 411 beige-selective genes from the left panel depicts comparison of expression ratio between WT-hJMJD1A-transduced and S265A-hJMJD1A-transduced im-scWATs (right). Changes are log 2 expression ratios of FPKM, as indicated in a color intensity scale. b , c qPCR analysis of beige-selective genes and Pparg in im-scWATs differentiated with or without ROS ( b ) or differentiated WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs ( c ). d H3K9me2 ChIP-qPCR on beige-selective genes using scWAT from age-matched WT (+/+) and Jmjd1a -null (−/−) mice placed at 4 °C for 1 week ( n = 4 per genotype group). e JMJD1A ChIP-qPCR on beige-selective genes during ROS-induced beige adipogenesis in im-scWATs. f ChIP-seq profiles for JMJD1A and PPARγ on Ucp1 , Cidea , and Ppara genomic regions in differentiated im-scWATs (beige cells) and im-BATs (BAT cells). g , h ChIP-qPCR showing isoproterenol (ISO) treatment increased JMJD1A recruitment in differentiated im-scWATs ( g ) and the decrease of H3K9me2 levels in beige-selective genes in differentiated beige adipocytes by ROS was blunted by Pro treatment ( h ). i JMJD1A ChIP-qPCR on beige-selective genes in scWAT of WT and Jmjd1a -S265A KI/KI mice following 1-week cold exposure ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 6). j ChIP-qPCR showing the decrease of H3K9me2 levels on indicated beige-selective genes during beige adipogenesis is impaired in S265A-hJMJD1A-transduced im-scWATs. The signal in day 0 of differentiation is set as 1. Data are mean ± s.e.m. of three technical replicates in a representative experiments performed at least three times ( b , c , e , g , h , j ). Student’s t test was performed for comparisons in d , i . * P
    Figure Legend Snippet: JMJD1A demethylates H3K9me2 on beige-selective genes. a RNA-seq heat map depicting expression ratio comparison between beige and white adipocytes, differentiated from im-scWATs under rosiglitazone (ROS) plus or minus condition (left). RNA-seq heat map of 411 beige-selective genes from the left panel depicts comparison of expression ratio between WT-hJMJD1A-transduced and S265A-hJMJD1A-transduced im-scWATs (right). Changes are log 2 expression ratios of FPKM, as indicated in a color intensity scale. b , c qPCR analysis of beige-selective genes and Pparg in im-scWATs differentiated with or without ROS ( b ) or differentiated WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs ( c ). d H3K9me2 ChIP-qPCR on beige-selective genes using scWAT from age-matched WT (+/+) and Jmjd1a -null (−/−) mice placed at 4 °C for 1 week ( n = 4 per genotype group). e JMJD1A ChIP-qPCR on beige-selective genes during ROS-induced beige adipogenesis in im-scWATs. f ChIP-seq profiles for JMJD1A and PPARγ on Ucp1 , Cidea , and Ppara genomic regions in differentiated im-scWATs (beige cells) and im-BATs (BAT cells). g , h ChIP-qPCR showing isoproterenol (ISO) treatment increased JMJD1A recruitment in differentiated im-scWATs ( g ) and the decrease of H3K9me2 levels in beige-selective genes in differentiated beige adipocytes by ROS was blunted by Pro treatment ( h ). i JMJD1A ChIP-qPCR on beige-selective genes in scWAT of WT and Jmjd1a -S265A KI/KI mice following 1-week cold exposure ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 6). j ChIP-qPCR showing the decrease of H3K9me2 levels on indicated beige-selective genes during beige adipogenesis is impaired in S265A-hJMJD1A-transduced im-scWATs. The signal in day 0 of differentiation is set as 1. Data are mean ± s.e.m. of three technical replicates in a representative experiments performed at least three times ( b , c , e , g , h , j ). Student’s t test was performed for comparisons in d , i . * P

    Techniques Used: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Mouse Assay

    Complementary mechanisms for thermogenic gene induction in acute and chronic cold stress via overlapping, but distinct mechanisms of JMJD1A. Brown fat cells mediate acute and robust thermogenic activation of Ucp1 , while scWAT-derived beige fat cells contribute to an adaptive response against chronic cold exposure (top). The acute response in BAT requires a BAR-dependent phosphorylation of JMJD1A that facilitates long-range enhancer-promoter interactions and stimulate thermogenic gene expressions, but this does not require the intrinsic H3K9me2 demethylation activity of JMJD1A (left bottom). The chronic adaptation in beigeing requires both phosphorylation-dependent chromatin recruitment and H3K9me2 demethylation activity of JMJD1A (right bottom). These histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT ensure an ordered transition between acute and chronic adaptation to cold stress. TXN transcription
    Figure Legend Snippet: Complementary mechanisms for thermogenic gene induction in acute and chronic cold stress via overlapping, but distinct mechanisms of JMJD1A. Brown fat cells mediate acute and robust thermogenic activation of Ucp1 , while scWAT-derived beige fat cells contribute to an adaptive response against chronic cold exposure (top). The acute response in BAT requires a BAR-dependent phosphorylation of JMJD1A that facilitates long-range enhancer-promoter interactions and stimulate thermogenic gene expressions, but this does not require the intrinsic H3K9me2 demethylation activity of JMJD1A (left bottom). The chronic adaptation in beigeing requires both phosphorylation-dependent chromatin recruitment and H3K9me2 demethylation activity of JMJD1A (right bottom). These histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT ensure an ordered transition between acute and chronic adaptation to cold stress. TXN transcription

    Techniques Used: Activation Assay, Derivative Assay, Activity Assay, Expressing

    Demethylation activity is pivotal for beige-selective gene inductions. a Schematic representation of the domain structure of human JMJD1A. Phosphorylation site at S265 and Fe(II) binding site at H1120 are shown. b OCR in im-scWATs stably expressing WT-hJMJD1A, S265D-hJMJD1A, or S265D-H1120Y-hJMJD1A treated sequentially with oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti) (left). Basal, maximum, and uncoupled respiration calculated from the left (right). Data are mean ± s.e.m. of three technical replicates in a representative experiment. Analysis of variance were performed, followed by Tukey’s post hoc comparison. * P
    Figure Legend Snippet: Demethylation activity is pivotal for beige-selective gene inductions. a Schematic representation of the domain structure of human JMJD1A. Phosphorylation site at S265 and Fe(II) binding site at H1120 are shown. b OCR in im-scWATs stably expressing WT-hJMJD1A, S265D-hJMJD1A, or S265D-H1120Y-hJMJD1A treated sequentially with oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti) (left). Basal, maximum, and uncoupled respiration calculated from the left (right). Data are mean ± s.e.m. of three technical replicates in a representative experiment. Analysis of variance were performed, followed by Tukey’s post hoc comparison. * P

    Techniques Used: Activity Assay, Binding Assay, Stable Transfection, Expressing

    Insulin resistance phenotype of Jmjd1a -S265A KI/KI mice. a Body weight changes. WT (+/+) and Jmjd1a -S265A KI/KI mice ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 8) were fed on HFD for 4 weeks at 30 °C, and then switched to 4 °C for 4 weeks. b , c Glucose tolerance test (GTT) ( b ) and insulin tolerance test (ITT) ( c ) in each genotype group mice fed on HFD after cold acclimation in a ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 7). d Assessment of insulin signaling as quantified by the phosphorylation of AKT-S473 in scWAT, BAT, or soleus from each genotype mice fed on HFD after cold acclimation presented in a . Data are mean ± s.e.m. ( a – c ) Student’s t test was performed for comparisons in a – c . * P
    Figure Legend Snippet: Insulin resistance phenotype of Jmjd1a -S265A KI/KI mice. a Body weight changes. WT (+/+) and Jmjd1a -S265A KI/KI mice ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 8) were fed on HFD for 4 weeks at 30 °C, and then switched to 4 °C for 4 weeks. b , c Glucose tolerance test (GTT) ( b ) and insulin tolerance test (ITT) ( c ) in each genotype group mice fed on HFD after cold acclimation in a ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 7). d Assessment of insulin signaling as quantified by the phosphorylation of AKT-S473 in scWAT, BAT, or soleus from each genotype mice fed on HFD after cold acclimation presented in a . Data are mean ± s.e.m. ( a – c ) Student’s t test was performed for comparisons in a – c . * P

    Techniques Used: Mouse Assay

    Ucp1 expression and H3K9me2 levels in adipose tissues and BAT thermogenic function in Jmjd1a -S265A KI/KI mice . a Ucp1 mRNA ( n = 3) (left) and protein levels detected by immunoblotting (IB) (right) in BAT, and scWAT of mice exposed to 4 °C for 6 h. b H3K9me2 immunoblotting using purified histones from adipose tissues of mice housed at RT. c H3K9me2 ChIP-qPCR in BAT, and scWAT of mice exposed to 4 °C for 6 h ( n = 3). d NE-induced Ucp1 mRNA levels in im-BATs stably expressing WT or indicated mutants of hJMJD1A. The relative quantity in im-BATs expressing WT-JMJD1A on day 8 before NE treatment (0 h) is defined as 1. e Ucp1 mRNA expressions ( n = 3) (left) and proteins (right) in BAT, and scWAT from mice exposed to 30 °C or 4 °C for 1 week. Uncropped images of the blots ( a , b , e . f , g H3K9me2 ChIP-qPCR in scWAT from mice placed at 4 °C for 1 week ( n = 4) ( f ) and in im-scWATs during beige adipogenesis (mean ± s.e.m. of three technical replicates) ( g ). h Bi-phasic Ucp1 expressions during cold exposure in BAT and scWAT. Mice were exposed to 4 °C for the indicated time, and Ucp1 mRNAs in BAT and scWATs were quantified by qPCR ( n = 3). The data were converted to copy number per ng of total RNA. i In BAT, Ucp 1 locus is in euchromatin (left), while in scWAT, it is in heterochromatin with H3K9me2 (right). In BAT, cold exposure leads to acute induction of Ucp1 mRNA through the mechanisms independent of H3K9me2 demethylation (left). In scWAT, H3K9me2 at Ucp1 gene locus needs to be removed for beige adipogenesis (right bottom). j Cold intolerance in Jmjd1a -S265A KI/KI mice ( n = 6 per genotype group). Shown is the body temperature of 9-week-old mice at different times after cold exposure (4 °C). k Impaired NE-induced activation of selective genes in BAT ( n = 6 per genotype group). l , m Reduced NE-induced mitochondrial respiration ( l ) and NE-induced glycerol release ( m ) in primary brown adipocytes from Jmjd1a -S265A KI/KI mice. Data are mean ± s.e.m. of five technical replicates ( l ) and three independent experiments ( m ). Data are mean ± s.e.m. a , c , e , f , j , k Student’s t test ( a , e , f , j , l , m ) or analysis of variance, followed by Tukey’s post hoc comparison ( k ) were performed for comparisons. * P
    Figure Legend Snippet: Ucp1 expression and H3K9me2 levels in adipose tissues and BAT thermogenic function in Jmjd1a -S265A KI/KI mice . a Ucp1 mRNA ( n = 3) (left) and protein levels detected by immunoblotting (IB) (right) in BAT, and scWAT of mice exposed to 4 °C for 6 h. b H3K9me2 immunoblotting using purified histones from adipose tissues of mice housed at RT. c H3K9me2 ChIP-qPCR in BAT, and scWAT of mice exposed to 4 °C for 6 h ( n = 3). d NE-induced Ucp1 mRNA levels in im-BATs stably expressing WT or indicated mutants of hJMJD1A. The relative quantity in im-BATs expressing WT-JMJD1A on day 8 before NE treatment (0 h) is defined as 1. e Ucp1 mRNA expressions ( n = 3) (left) and proteins (right) in BAT, and scWAT from mice exposed to 30 °C or 4 °C for 1 week. Uncropped images of the blots ( a , b , e . f , g H3K9me2 ChIP-qPCR in scWAT from mice placed at 4 °C for 1 week ( n = 4) ( f ) and in im-scWATs during beige adipogenesis (mean ± s.e.m. of three technical replicates) ( g ). h Bi-phasic Ucp1 expressions during cold exposure in BAT and scWAT. Mice were exposed to 4 °C for the indicated time, and Ucp1 mRNAs in BAT and scWATs were quantified by qPCR ( n = 3). The data were converted to copy number per ng of total RNA. i In BAT, Ucp 1 locus is in euchromatin (left), while in scWAT, it is in heterochromatin with H3K9me2 (right). In BAT, cold exposure leads to acute induction of Ucp1 mRNA through the mechanisms independent of H3K9me2 demethylation (left). In scWAT, H3K9me2 at Ucp1 gene locus needs to be removed for beige adipogenesis (right bottom). j Cold intolerance in Jmjd1a -S265A KI/KI mice ( n = 6 per genotype group). Shown is the body temperature of 9-week-old mice at different times after cold exposure (4 °C). k Impaired NE-induced activation of selective genes in BAT ( n = 6 per genotype group). l , m Reduced NE-induced mitochondrial respiration ( l ) and NE-induced glycerol release ( m ) in primary brown adipocytes from Jmjd1a -S265A KI/KI mice. Data are mean ± s.e.m. of five technical replicates ( l ) and three independent experiments ( m ). Data are mean ± s.e.m. a , c , e , f , j , k Student’s t test ( a , e , f , j , l , m ) or analysis of variance, followed by Tukey’s post hoc comparison ( k ) were performed for comparisons. * P

    Techniques Used: Expressing, Mouse Assay, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Activation Assay

    Phospho-S265 JMJD1A induces beige biogenesis. a qPCR analysis demonstrates decreased expression of beige-selective genes in scWAT from Jmjd1a -S265A KI/KI mice exposed to 30 °C or 4 °C for 1 week ( n = 5 per genotype group). b Immunoblot analysis of UCP1 and PPARγ in tissue homogenates of scWAT from mice presented in a . c Haematoxylin and eosin (H E) and UCP1 staining sections of scWAT from WT and Jmjd1a -S265A KI/KI mice exposed to chronic cold exposure (4 °C for 1 week) (scale bar, 100 μm). d NE-induced oxygen consumption rate (OCR) in mice exposed to chronic cold exposure (4 °C for 4 weeks) ( n = 7 per genotype group) (left). OCR before and 30 min after NE treatment are analyzed (right) ( n = 7). e OCR of scWAT from mice exposed to 30 °C or 4 °C for 1 week ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 4). Data are mean ± s.e.m. a , d , e Analysis of variance were performed followed by Tukey’s post hoc comparison in a . Student’s t test was performed for comparisons in d , e . * P
    Figure Legend Snippet: Phospho-S265 JMJD1A induces beige biogenesis. a qPCR analysis demonstrates decreased expression of beige-selective genes in scWAT from Jmjd1a -S265A KI/KI mice exposed to 30 °C or 4 °C for 1 week ( n = 5 per genotype group). b Immunoblot analysis of UCP1 and PPARγ in tissue homogenates of scWAT from mice presented in a . c Haematoxylin and eosin (H E) and UCP1 staining sections of scWAT from WT and Jmjd1a -S265A KI/KI mice exposed to chronic cold exposure (4 °C for 1 week) (scale bar, 100 μm). d NE-induced oxygen consumption rate (OCR) in mice exposed to chronic cold exposure (4 °C for 4 weeks) ( n = 7 per genotype group) (left). OCR before and 30 min after NE treatment are analyzed (right) ( n = 7). e OCR of scWAT from mice exposed to 30 °C or 4 °C for 1 week ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 4). Data are mean ± s.e.m. a , d , e Analysis of variance were performed followed by Tukey’s post hoc comparison in a . Student’s t test was performed for comparisons in d , e . * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Staining

    β-Adrenergic signal is required for the induction of beige-selective genes mediated by PPARγ ligand. a WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs were differentiated for beige adipogenesis in the presence or absence of propranolol (Pro), as schematically illustrated (top), and ORO staining was performed (bottom). b Whole-cell lysates (WCL) from WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs differentiated under Pro (100 nM) plus or minus condition were subjected to immunoprecipitation (IP) with anti-V5 antibody, followed by immunoblot (IB) analysis with anti-P-JMJD1A (pSer265) antibody. c qPCR analysis of beige-selective genes and general adipogenic genes in WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs under Pro plus or minus condition (mean ± s.e.m. of three technical replicates). d Immunoblotting with anti-UCP1 or anti-total OXPHOS antibodies cocktail using WCL from indicated viral transduced im-scWATs, differentiated under Pro plus or minus condition. e OCRs (basal, maximum, and uncoupled) of WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs, differentiated under Pro plus or minus condition. Data are mean ± s.e.m. of five technical replicates. f , g Immunoblotting with anti-PRDM16, anti-P-JMJD1A, anti-V5, anti-PPARγ, or anti-PGC1α antibody following immunoprecipitation with anti-PRDM16 antibody ( f ) or with anti-V5 antibody for JMJD1A ( g ), from WCL of differentiated WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs. Uncropped images of the blots ( b , d , f , g . Analysis of variance was performed, followed by Tukey’s post hoc comparison in e . * P
    Figure Legend Snippet: β-Adrenergic signal is required for the induction of beige-selective genes mediated by PPARγ ligand. a WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs were differentiated for beige adipogenesis in the presence or absence of propranolol (Pro), as schematically illustrated (top), and ORO staining was performed (bottom). b Whole-cell lysates (WCL) from WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs differentiated under Pro (100 nM) plus or minus condition were subjected to immunoprecipitation (IP) with anti-V5 antibody, followed by immunoblot (IB) analysis with anti-P-JMJD1A (pSer265) antibody. c qPCR analysis of beige-selective genes and general adipogenic genes in WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs under Pro plus or minus condition (mean ± s.e.m. of three technical replicates). d Immunoblotting with anti-UCP1 or anti-total OXPHOS antibodies cocktail using WCL from indicated viral transduced im-scWATs, differentiated under Pro plus or minus condition. e OCRs (basal, maximum, and uncoupled) of WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs, differentiated under Pro plus or minus condition. Data are mean ± s.e.m. of five technical replicates. f , g Immunoblotting with anti-PRDM16, anti-P-JMJD1A, anti-V5, anti-PPARγ, or anti-PGC1α antibody following immunoprecipitation with anti-PRDM16 antibody ( f ) or with anti-V5 antibody for JMJD1A ( g ), from WCL of differentiated WT-hJMJD1A-transduced or S265A-hJMJD1A-transduced im-scWATs. Uncropped images of the blots ( b , d , f , g . Analysis of variance was performed, followed by Tukey’s post hoc comparison in e . * P

    Techniques Used: Staining, Immunoprecipitation, Real-time Polymerase Chain Reaction

    P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P
    Figure Legend Snippet: P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Techniques Used: Knock-In, Staining, Real-time Polymerase Chain Reaction

    30) Product Images from "Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch"

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03868-8

    P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P
    Figure Legend Snippet: P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Techniques Used: Knock-In, Staining, Real-time Polymerase Chain Reaction

    31) Product Images from "Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch"

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03868-8

    P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P
    Figure Legend Snippet: P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Techniques Used: Knock-In, Staining, Real-time Polymerase Chain Reaction

    32) Product Images from "The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals"

    Article Title: The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35499-w

    Southern blot analysis of MUC2 PTS-TR2 integrity shows variable number of tandem repeats. Southern blot using SMUC41 probe on small-scale purified RP13-870H17 BAC DNA (5–10 μg/lane). Expected bands are indicated by * and sizes are given in kilo bases (kb) ( a ) Sac I /Hind III-digested, Lane 1 , and Apa I-digested, Lane 2 , original RP13-870H17 BAC clone showed more bands than expected. ( b ) Apa I digest of different clones, Lanes 1 – 5 , of the BAC showed PTS-TR2 variants from 98TR, Lane 1 , down to 8TR, Lane 2 . ( c .
    Figure Legend Snippet: Southern blot analysis of MUC2 PTS-TR2 integrity shows variable number of tandem repeats. Southern blot using SMUC41 probe on small-scale purified RP13-870H17 BAC DNA (5–10 μg/lane). Expected bands are indicated by * and sizes are given in kilo bases (kb) ( a ) Sac I /Hind III-digested, Lane 1 , and Apa I-digested, Lane 2 , original RP13-870H17 BAC clone showed more bands than expected. ( b ) Apa I digest of different clones, Lanes 1 – 5 , of the BAC showed PTS-TR2 variants from 98TR, Lane 1 , down to 8TR, Lane 2 . ( c .

    Techniques Used: Southern Blot, Purification, BAC Assay, Clone Assay

    33) Product Images from "Genotyping 1000 yeast strains by next-generation sequencing"

    Article Title: Genotyping 1000 yeast strains by next-generation sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-90

    Quality control of fragmented DNA. ( A ) Bioanalyzer results from three DNA samples fragmented either in glass tubes with a Covaris DNA shearing device (Duty cycle 10%, Intensity 4.5, Cycles per burst 200, Time 120 s), or in PCR strips with a Bandelin sonicator (2 times 4 min). ( B ) 1.5% agarose gel loaded with 22 samples fragmented by Bandelin sonication. The size distribution is very narrow (major peak between 100–300 bp) and has an acceptable reproducibility.
    Figure Legend Snippet: Quality control of fragmented DNA. ( A ) Bioanalyzer results from three DNA samples fragmented either in glass tubes with a Covaris DNA shearing device (Duty cycle 10%, Intensity 4.5, Cycles per burst 200, Time 120 s), or in PCR strips with a Bandelin sonicator (2 times 4 min). ( B ) 1.5% agarose gel loaded with 22 samples fragmented by Bandelin sonication. The size distribution is very narrow (major peak between 100–300 bp) and has an acceptable reproducibility.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sonication

    Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.
    Figure Legend Snippet: Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.

    Techniques Used: DNA Extraction, Sonication, Polymerase Chain Reaction, Ligation, Incubation

    Comparison of coverage homogeneity and GC bias between different techniques. ( A ) The distribution of per-base depths was calculated (with only uniquely aligned reads) for our heat-inactivation protocol using either Covaris fragmentation (black) or Bandelin fragmentation (red), and is comparable to the standard library preparation, in which Covaris fragmentation was used in combination with SPRI cleanups (blue). ( B ) The GC bias is low for all compared techniques, as depicted on the right, with a slightly larger bias for the heat-inactivation protocols (using a mean depth of 200 bp bins, LOESS smooth with span = 0.3).
    Figure Legend Snippet: Comparison of coverage homogeneity and GC bias between different techniques. ( A ) The distribution of per-base depths was calculated (with only uniquely aligned reads) for our heat-inactivation protocol using either Covaris fragmentation (black) or Bandelin fragmentation (red), and is comparable to the standard library preparation, in which Covaris fragmentation was used in combination with SPRI cleanups (blue). ( B ) The GC bias is low for all compared techniques, as depicted on the right, with a slightly larger bias for the heat-inactivation protocols (using a mean depth of 200 bp bins, LOESS smooth with span = 0.3).

    Techniques Used:

    34) Product Images from "A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454"

    Article Title: A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

    Journal: Genome Biology

    doi: 10.1186/gb-2010-11-2-r15

    Robust, optimized plate-based acoustic shearing of genomic DNA . (a) Effect of time on shearing profile. Agilent Bioanalyzer traces of 3 μg human genomic DNA (Promega) diluted in 100 μl, aliquoted into an ABI PRISM™ Optical Reaction plate and sheared in the Covaris™ E210 under standard plate conditions (duty cycle = 5, intensity = 5, cycles per burst = 500) for increasing amounts of time (n = 3 for each timepoint). (b) Incomplete shears recovered by re-shearing. (i) Average shearing distribution (n = 27) of samples sheared for 100 seconds under standard conditions. (ii) An example of incomplete shearing seen in three attempts under standard conditions. (iii) Resultant fragment pattern after reshearing from (ii) with standard conditions. Each shear profile signal is plotted normalized to the maximum ladder fluorescence for the Bioanalyzer chip upon which the sample was run. (c) Dual high and low cutoff size-selection using para-magnetic beads (SPRI). Human genomic DNA (3 μg) was sheared under standard conditions, producing fragments ranging in size from less than 100 bp to approximately 4 kb (i). This shear product then underwent a 0.5× Solid Phase Reversible Immobilization (SPRI) reaction in which high molecular weight fragments were preferentially bound (ii). The supernatant was removed to a second tube and underwent a second 0.7× SPRI reaction where fragments below 300 bp were removed in the supernatant (iii). Fragments in the desired size range of 300 to 1,000 bp were eluted from the beads (iv).
    Figure Legend Snippet: Robust, optimized plate-based acoustic shearing of genomic DNA . (a) Effect of time on shearing profile. Agilent Bioanalyzer traces of 3 μg human genomic DNA (Promega) diluted in 100 μl, aliquoted into an ABI PRISM™ Optical Reaction plate and sheared in the Covaris™ E210 under standard plate conditions (duty cycle = 5, intensity = 5, cycles per burst = 500) for increasing amounts of time (n = 3 for each timepoint). (b) Incomplete shears recovered by re-shearing. (i) Average shearing distribution (n = 27) of samples sheared for 100 seconds under standard conditions. (ii) An example of incomplete shearing seen in three attempts under standard conditions. (iii) Resultant fragment pattern after reshearing from (ii) with standard conditions. Each shear profile signal is plotted normalized to the maximum ladder fluorescence for the Bioanalyzer chip upon which the sample was run. (c) Dual high and low cutoff size-selection using para-magnetic beads (SPRI). Human genomic DNA (3 μg) was sheared under standard conditions, producing fragments ranging in size from less than 100 bp to approximately 4 kb (i). This shear product then underwent a 0.5× Solid Phase Reversible Immobilization (SPRI) reaction in which high molecular weight fragments were preferentially bound (ii). The supernatant was removed to a second tube and underwent a second 0.7× SPRI reaction where fragments below 300 bp were removed in the supernatant (iii). Fragments in the desired size range of 300 to 1,000 bp were eluted from the beads (iv).

    Techniques Used: Fluorescence, Chromatin Immunoprecipitation, Selection, Magnetic Beads, Molecular Weight

    35) Product Images from "A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome"

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112950

    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.
    Figure Legend Snippet: Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Techniques Used: Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    36) Product Images from "Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis"

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis

    Journal: Scientific Reports

    doi: 10.1038/srep25310

    DNA damage-induced mutagenesis and EasyMF sequencing library preparation. (a) Schematic diagram of mutation frequency analysis in pSP189 using traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. The undamaged or damaged (220 J/m 2 UVC) pSP189 plasmid was transfected into the indicated siRNA-treated 293T cells. Mutation frequency in pSP189 was determined after it was recovered from 293T cells through both traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. (b) The diagram of “EasyMF”. DNA was sheared into fragments shorter than half the length of the sequencing read and circularized, then used for RCA. Next, the amplified DNA were taken to prepare standard NGS libraries. The real mutation will appear in each tandem repeats (red dots), conversely, errors resulted from the RCA or NGS library preparation only happen in some of repeats (blue dots) randomly. Since the size of sheared DNA fragments shorter than the length of a single PE read, the original DNA will be sequenced at least twice in a pair of PE reads independently to eliminate PCR and sequencing errors. The consensus sequence (CS) can be determined by align read 1 and read 2 in one pair of PE reads, and the site of CS will be given with high quality scores if it is supported by at least twice, and it will be given with the lowest quality scores if it is supported by only once.
    Figure Legend Snippet: DNA damage-induced mutagenesis and EasyMF sequencing library preparation. (a) Schematic diagram of mutation frequency analysis in pSP189 using traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. The undamaged or damaged (220 J/m 2 UVC) pSP189 plasmid was transfected into the indicated siRNA-treated 293T cells. Mutation frequency in pSP189 was determined after it was recovered from 293T cells through both traditional supF shuttle vector-based mutagenesis assay (TSMA) and “EasyMF”. (b) The diagram of “EasyMF”. DNA was sheared into fragments shorter than half the length of the sequencing read and circularized, then used for RCA. Next, the amplified DNA were taken to prepare standard NGS libraries. The real mutation will appear in each tandem repeats (red dots), conversely, errors resulted from the RCA or NGS library preparation only happen in some of repeats (blue dots) randomly. Since the size of sheared DNA fragments shorter than the length of a single PE read, the original DNA will be sequenced at least twice in a pair of PE reads independently to eliminate PCR and sequencing errors. The consensus sequence (CS) can be determined by align read 1 and read 2 in one pair of PE reads, and the site of CS will be given with high quality scores if it is supported by at least twice, and it will be given with the lowest quality scores if it is supported by only once.

    Techniques Used: Mutagenesis, Sequencing, Plasmid Preparation, Transfection, Amplification, Next-Generation Sequencing, Polymerase Chain Reaction

    37) Product Images from "Cystic renal‐epithelial derived induced pluripotent stem cells from polycystic kidney disease patients, et al. Cystic renal‐epithelial derived induced pluripotent stem cells from polycystic kidney disease patients"

    Article Title: Cystic renal‐epithelial derived induced pluripotent stem cells from polycystic kidney disease patients, et al. Cystic renal‐epithelial derived induced pluripotent stem cells from polycystic kidney disease patients

    Journal: Stem Cells Translational Medicine

    doi: 10.1002/sctm.18-0283

    Germline and somatic mutation analysis cyst derived tubular epithelial cells (TECs). A, Heterozygous germline mutations in patient 6 and patient 9 present in TECs from 3 cysts result in a frameshift. B, MeD‐seq analysis of PKD1 showing read‐count scores per LpnPI site, revealing no increased DNA methylation in TECs obtained from cyst lining epithelium (promoter shown in blue). C, mRNA expression levels of PKD1 and PKD2 in TECs and iPSCs (qRT‐PCR), normalized by the average of two housekeeping genes; actin and GAPDH, error bars represent the SD. D, Somatic mutations observed by whole‐exome sequencing comparing cysts of the same patient
    Figure Legend Snippet: Germline and somatic mutation analysis cyst derived tubular epithelial cells (TECs). A, Heterozygous germline mutations in patient 6 and patient 9 present in TECs from 3 cysts result in a frameshift. B, MeD‐seq analysis of PKD1 showing read‐count scores per LpnPI site, revealing no increased DNA methylation in TECs obtained from cyst lining epithelium (promoter shown in blue). C, mRNA expression levels of PKD1 and PKD2 in TECs and iPSCs (qRT‐PCR), normalized by the average of two housekeeping genes; actin and GAPDH, error bars represent the SD. D, Somatic mutations observed by whole‐exome sequencing comparing cysts of the same patient

    Techniques Used: Mutagenesis, Derivative Assay, DNA Methylation Assay, Expressing, Quantitative RT-PCR, Sequencing

    38) Product Images from "Phylogenomic Resolution of the Cetacean Tree of Life Using Target Sequence Capture"

    Article Title: Phylogenomic Resolution of the Cetacean Tree of Life Using Target Sequence Capture

    Journal: Systematic Biology

    doi: 10.1093/sysbio/syz068

    The best concatenated RAxML maximum likelihood tree derived from Dataset A using 3191 partitions of each protein-coding gene and 6,527,596 bp (lnL = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$-$\end{document} 24,850,884.943687). The phylogenetic relationships of Delphininae are shown more clearly in the box on the left. All concatenated RAxML and Bayesian analyses using Dataset A retrieved the same topology. All nodes have 100% BS values (RAxML) or 1.0 PP (Exabayes) with the exception of the numbered nodes represented by red dots. These nodes have BS values and PP shown in the table in the upper left. The ASTRAL species tree topology only differed from the concatenated topology at three nodes shown in blue. Taxa in bold are those with data derived from previously existing genomes, transcriptomes, or GenBank sequences. Illustrations are by Carl Buell and represent (top to bottom) T. truncatus (common bottlenose dolphin), Feresa attenuata (pygmy killer whale), L. albirostris (white-beaked dolphin), Inia geoffrensis (Amazon river dolphin), Mesoplodon layardii (strap-toothed whale), Kogia sima (dwarf sperm whale), B. bonaerensis (Antarctic minke whale), and B. taurus (domestic cow).
    Figure Legend Snippet: The best concatenated RAxML maximum likelihood tree derived from Dataset A using 3191 partitions of each protein-coding gene and 6,527,596 bp (lnL = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$-$\end{document} 24,850,884.943687). The phylogenetic relationships of Delphininae are shown more clearly in the box on the left. All concatenated RAxML and Bayesian analyses using Dataset A retrieved the same topology. All nodes have 100% BS values (RAxML) or 1.0 PP (Exabayes) with the exception of the numbered nodes represented by red dots. These nodes have BS values and PP shown in the table in the upper left. The ASTRAL species tree topology only differed from the concatenated topology at three nodes shown in blue. Taxa in bold are those with data derived from previously existing genomes, transcriptomes, or GenBank sequences. Illustrations are by Carl Buell and represent (top to bottom) T. truncatus (common bottlenose dolphin), Feresa attenuata (pygmy killer whale), L. albirostris (white-beaked dolphin), Inia geoffrensis (Amazon river dolphin), Mesoplodon layardii (strap-toothed whale), Kogia sima (dwarf sperm whale), B. bonaerensis (Antarctic minke whale), and B. taurus (domestic cow).

    Techniques Used: Derivative Assay

    Infinite-sites plots showing the estimated posterior mean times in Ma ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$x$\end{document} -axis) plotted against the estimated posterior confidence interval (CI) widths in Ma ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y$\end{document} -axis) for the AR model for both data sets (all data, 10 genes SortaDate) using the three different partition schemes, three partitions (a, a’), six partitions (b, b’), and 10 partitions (c, c’). The solid line represents the regression line including the root and the dotted line represents the regression line excluding the root. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$R^{2}$\end{document} is the coefficient of determination for each comparison, whereas below each are the equations of the regression lines with and without the root.
    Figure Legend Snippet: Infinite-sites plots showing the estimated posterior mean times in Ma ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$x$\end{document} -axis) plotted against the estimated posterior confidence interval (CI) widths in Ma ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y$\end{document} -axis) for the AR model for both data sets (all data, 10 genes SortaDate) using the three different partition schemes, three partitions (a, a’), six partitions (b, b’), and 10 partitions (c, c’). The solid line represents the regression line including the root and the dotted line represents the regression line excluding the root. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$R^{2}$\end{document} is the coefficient of determination for each comparison, whereas below each are the equations of the regression lines with and without the root.

    Techniques Used:

    Scatterplot of the estimated posterior mean times (and 95% confidence intervals) for the six-partition scheme of both AR (a) and IR (b) models for the SortaDate data set ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$x$\end{document} -axis) against all data ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y$\end{document} -axis).
    Figure Legend Snippet: Scatterplot of the estimated posterior mean times (and 95% confidence intervals) for the six-partition scheme of both AR (a) and IR (b) models for the SortaDate data set ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$x$\end{document} -axis) against all data ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y$\end{document} -axis).

    Techniques Used:

    39) Product Images from "Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq)"

    Article Title: Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq)

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7768-0_12

    Size distribution analysis of a NOMe-seq sample and library Shown is a Bioanalyzer trace, obtained using an Agilent 2100 Bioanalyzer instrument and an Agilent High Sensitivity DNA chip, of the DNA after M.CviPI treatment and fragmentation using a Covaris S220 sonicator (a) and of the resultant NOMe-seq library (b) . The leftmost and rightmost peaks (labeled 43 and 113) are size markers of 35 bp and 10380 bp, respectively. The average length of the fragmented DNA is calculated to be 150 bp whereas the average length of the library fragments is calculated to be 280 bp. (c) For comparison to the Bioanalyzer traces, the gel images of the fragmented DNA and the NOMe-seq library are also shown.
    Figure Legend Snippet: Size distribution analysis of a NOMe-seq sample and library Shown is a Bioanalyzer trace, obtained using an Agilent 2100 Bioanalyzer instrument and an Agilent High Sensitivity DNA chip, of the DNA after M.CviPI treatment and fragmentation using a Covaris S220 sonicator (a) and of the resultant NOMe-seq library (b) . The leftmost and rightmost peaks (labeled 43 and 113) are size markers of 35 bp and 10380 bp, respectively. The average length of the fragmented DNA is calculated to be 150 bp whereas the average length of the library fragments is calculated to be 280 bp. (c) For comparison to the Bioanalyzer traces, the gel images of the fragmented DNA and the NOMe-seq library are also shown.

    Techniques Used: Chromatin Immunoprecipitation, Labeling

    40) Product Images from "Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes"

    Article Title: Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078575

    A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.
    Figure Legend Snippet: A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.

    Techniques Used: Concentration Assay

    Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.
    Figure Legend Snippet: Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.

    Techniques Used: Concentration Assay, Sequencing

    Related Articles

    Sonication:

    Article Title: Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages
    Article Snippet: .. For CDK9, nuclei were sonicated with Covaris S220 Ultrasonicator according to manufacturer’s instructions (130 μl shearing buffer, 200 cycles/burst, 120 s, DF 10). .. Lysates were cleared by centrifugation at 14,000*g, 20 min, 4°C, and then incubated with normal rabbit IgG (Santa Cruz Biotech, sc-2027x), BRD4 (Abcam, ab84776 and Bethyl Laboratories, A3001-985A100), MED12 (Bethyl Laboratories, A300-774A), MED1 (Bethyl Laboratories, A300-793), p300 (Santa Cruz Biotech, sc-585X), Anti-AcH4 (Millipore, 06–866), Anti-AcH4K12 (Millipore, 07–595), Anti-AcH4K5 (Millipore, 07–327) and 40 μl of 50% protein A/G plus agarose (Santa Cruz Biotech, sc-2003) per reaction at 4°C ON.

    Article Title: Loss of Tet enzymes compromises proper differentiation of embryonic stem cells
    Article Snippet: .. 2 μg of genomic DNA isolated from day 10 EBs was sonicated to 300bp fragments using a Covaris S220 ultrasonicator (Covaris). .. One μg of purified adapter ligated DNA was used for pooled immunoprecipitations using a polyclonal 5mC-specific antibody (Active Motif, 39791).

    Purification:

    Article Title: Functional and evolutionary insights from the Ciona notochord transcriptome
    Article Snippet: .. Purified RNA (2.5 μl) was used in half-reactions following their standard protocol. cDNA samples were fragmented with a Covaris S220 Ultrasonicator using the recommended settings to obtain an average fragment length of 300 bp and the NEBNext DNA Library Prep Master Mix set for Illumina from New England BioLabs (NEB) was used for library construction. .. The resulting libraries were quality checked with an Agilent 2100 Bioanalyzer and quantified by RT-PCR.

    Article Title: Pancreatic islet chromatin accessibility and conformation reveals distal enhancer networks of type 2 diabetes risk
    Article Snippet: .. DNA was purified with phenol-chloroform extraction and ethanol precipitation, followed by fragmentation to 300–500 bp with the Covaris S220 ultrasonicator. .. Ligation products were enriched with Dynabeads My One T1 Streptavidin beads (Life Technologies).

    Isolation:

    Article Title: Loss of Tet enzymes compromises proper differentiation of embryonic stem cells
    Article Snippet: .. 2 μg of genomic DNA isolated from day 10 EBs was sonicated to 300bp fragments using a Covaris S220 ultrasonicator (Covaris). .. One μg of purified adapter ligated DNA was used for pooled immunoprecipitations using a polyclonal 5mC-specific antibody (Active Motif, 39791).

    Ethanol Precipitation:

    Article Title: Pancreatic islet chromatin accessibility and conformation reveals distal enhancer networks of type 2 diabetes risk
    Article Snippet: .. DNA was purified with phenol-chloroform extraction and ethanol precipitation, followed by fragmentation to 300–500 bp with the Covaris S220 ultrasonicator. .. Ligation products were enriched with Dynabeads My One T1 Streptavidin beads (Life Technologies).

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    Covaris um 226 genomic dna
    Colonial characteristic and microscopic morphology of Bipolaris papendorfii UM 226. The surface (A) and reverse (B) colony morphology of B. papendorfii <t>UM</t> 226 after being cultured for 7 days. Light micrograph showing (C) typical zig-zag conidiophore with several conidia and (D) conidia with three pseudoseptates (×400 magnification, bars 20 µm). Scanning electron micrograph showing (E) zig-zag conidiophores with verruculose walled conidia (×3,065 magnification, bars 10 µm). This figure is available in black and white in print and in colour at <t>DNA</t> Research online.
    Um 226 Genomic Dna, supplied by Covaris, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covaris bulk input dna
    Single-Cell RC-Seq Workflow (A) NeuN + hippocampal nuclei were first purified by FACS (see also Figure S1 ). (B) Nuclei were then picked using a self-contained microscope and micromanipulator. (C) <t>DNA</t> was extracted from nuclei and subjected to linear WGA, followed by exponential PCR in two separate reactions for each nucleus, using different enzymes. (D) Exponential WGA products for each nucleus were combined, used to prepare <t>Illumina</t> libraries, and analyzed via WGS to assess genome coverage and possible amplification biases. (E) Libraries prepared in (D) were enriched via hybridization to L1-Ta LNA probes. (F) Enriched libraries were sequenced with 2 × 150-mer Illumina reads and analyzed to identify novel L1 integration sites (see also Figure S2 ).
    Bulk Input Dna, supplied by Covaris, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covaris droplet cirseq library preparation genomic dna
    Error rate and mutation types of <t>Droplet-CirSeq.</t> a Error rate of Droplet-CirSeq and Cir-seq. The “1X allele” represents the bases that were supported at least by one circularized <t>DNA,</t> while the “2X allele” represents the bases that were supported by at least two different circularized DNAs. The error rate of Droplet-CirSeq was 5.23 X 10 -5 (±1.54 X 10 -5 ) at the “1X allele” criterion and 3.71 X 10 -6 (±2.34 X 10 -7 ) at the “2X allele” criterion. b Mutation types of Droplet-CirSeq. The error rates of most of the mutation types were lower than 3.00 X 10 -6 , but the error rates for the transitions C= > T and G= > A were almost one order of magnitude higher than the other types, and the other two transitions, A= > G and T= > C, also showed high error rates. The mutation pattern of the “2X allele” showed the same pattern as with the “1X allele”
    Droplet Cirseq Library Preparation Genomic Dna, supplied by Covaris, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covaris cyanophage genomic dna
    Proposed role of 2-oxoglutarate (2OG) during <t>cyanophage</t> infection. A. In uninfected cyanobacteria, nitrogen limitation causes 2OG to accumulate, leading to 2OG-dependent binding of NtcA to promoters of nitrogen-stress genes, resulting in their expression. B. Phage infection draws down cellular nitrogen causing N-stress and likely leading to 2OG accumulation. Several cyanophage-encoded enzymes (in bold) suggest that increased 2OG may facilitate phage infection. First, a putative phytanoyl-CoA dioxygenase may convert 2OG to succinate, a major electron donor to respiratory electron transport in cyanobacteria ( Cooley and Vermaas, 2001 ) thus potentially generating energy for the infection process. Second, 2OG-dependent dioxygenase [2OG-Fe(II)] superfamily proteins may function in cyanophage <t>DNA</t> repair ( Weigele et al ., 2007 ). Third, cyanophage genomes have multiple NtcA promoters driving genes encoding diverse functions – possibly exploiting the host NtcA-driven N-stress response system.
    Cyanophage Genomic Dna, supplied by Covaris, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Colonial characteristic and microscopic morphology of Bipolaris papendorfii UM 226. The surface (A) and reverse (B) colony morphology of B. papendorfii UM 226 after being cultured for 7 days. Light micrograph showing (C) typical zig-zag conidiophore with several conidia and (D) conidia with three pseudoseptates (×400 magnification, bars 20 µm). Scanning electron micrograph showing (E) zig-zag conidiophores with verruculose walled conidia (×3,065 magnification, bars 10 µm). This figure is available in black and white in print and in colour at DNA Research online.

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

    Article Title: Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

    doi: 10.1093/dnares/dsv007

    Figure Lengend Snippet: Colonial characteristic and microscopic morphology of Bipolaris papendorfii UM 226. The surface (A) and reverse (B) colony morphology of B. papendorfii UM 226 after being cultured for 7 days. Light micrograph showing (C) typical zig-zag conidiophore with several conidia and (D) conidia with three pseudoseptates (×400 magnification, bars 20 µm). Scanning electron micrograph showing (E) zig-zag conidiophores with verruculose walled conidia (×3,065 magnification, bars 10 µm). This figure is available in black and white in print and in colour at DNA Research online.

    Article Snippet: UM 226 genomic DNA was sheared into smaller fragments by Covaris S/E210 or Bioruptor.

    Techniques: Cell Culture

    MAT1-2 gene of Bipolaris papendorfii UM 226. (A) Schematic representation of major open reading frames (ORFs) identified in the MAT regions of B. papendorfii UM 226 and Cochliobolus heterostrophus C4. Numbers are in kilobases. (B) Bayesian phylogram generated based on MAT1-2 nucleotide sequences. The tree is rooted with Alternaria alternata as outgroup. Numbers on the nodes indicate Bayesian posterior probability based on 100 sampling frequency for a total of 150,000 generations. This figure is available in black and white in print and in colour at DNA Research online.

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

    Article Title: Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

    doi: 10.1093/dnares/dsv007

    Figure Lengend Snippet: MAT1-2 gene of Bipolaris papendorfii UM 226. (A) Schematic representation of major open reading frames (ORFs) identified in the MAT regions of B. papendorfii UM 226 and Cochliobolus heterostrophus C4. Numbers are in kilobases. (B) Bayesian phylogram generated based on MAT1-2 nucleotide sequences. The tree is rooted with Alternaria alternata as outgroup. Numbers on the nodes indicate Bayesian posterior probability based on 100 sampling frequency for a total of 150,000 generations. This figure is available in black and white in print and in colour at DNA Research online.

    Article Snippet: UM 226 genomic DNA was sheared into smaller fragments by Covaris S/E210 or Bioruptor.

    Techniques: Generated, Sampling

    Single-Cell RC-Seq Workflow (A) NeuN + hippocampal nuclei were first purified by FACS (see also Figure S1 ). (B) Nuclei were then picked using a self-contained microscope and micromanipulator. (C) DNA was extracted from nuclei and subjected to linear WGA, followed by exponential PCR in two separate reactions for each nucleus, using different enzymes. (D) Exponential WGA products for each nucleus were combined, used to prepare Illumina libraries, and analyzed via WGS to assess genome coverage and possible amplification biases. (E) Libraries prepared in (D) were enriched via hybridization to L1-Ta LNA probes. (F) Enriched libraries were sequenced with 2 × 150-mer Illumina reads and analyzed to identify novel L1 integration sites (see also Figure S2 ).

    Journal: Cell

    Article Title: Ubiquitous L1 Mosaicism in Hippocampal Neurons

    doi: 10.1016/j.cell.2015.03.026

    Figure Lengend Snippet: Single-Cell RC-Seq Workflow (A) NeuN + hippocampal nuclei were first purified by FACS (see also Figure S1 ). (B) Nuclei were then picked using a self-contained microscope and micromanipulator. (C) DNA was extracted from nuclei and subjected to linear WGA, followed by exponential PCR in two separate reactions for each nucleus, using different enzymes. (D) Exponential WGA products for each nucleus were combined, used to prepare Illumina libraries, and analyzed via WGS to assess genome coverage and possible amplification biases. (E) Libraries prepared in (D) were enriched via hybridization to L1-Ta LNA probes. (F) Enriched libraries were sequenced with 2 × 150-mer Illumina reads and analyzed to identify novel L1 integration sites (see also Figure S2 ).

    Article Snippet: Illumina Library Construction Multiplexed Illumina libraries were prepared from bulk input DNA following a previous method ( ) with the following modifications: 1 μg input DNA quantified using a Qubit HS DNA Fluorometer was sonicated using a Covaris M220 (Covaris Woburn, Massachusetts) with the following settings: peak power 50, duty factor 20, cycles per burst 200, and time 120 sec. DNA was end repaired, A-tailed, and ligated following the Illumina TruSeq library preparation protocol.

    Techniques: Purification, FACS, Microscopy, Whole Genome Amplification, Polymerase Chain Reaction, Amplification, Hybridization

    Error rate and mutation types of Droplet-CirSeq. a Error rate of Droplet-CirSeq and Cir-seq. The “1X allele” represents the bases that were supported at least by one circularized DNA, while the “2X allele” represents the bases that were supported by at least two different circularized DNAs. The error rate of Droplet-CirSeq was 5.23 X 10 -5 (±1.54 X 10 -5 ) at the “1X allele” criterion and 3.71 X 10 -6 (±2.34 X 10 -7 ) at the “2X allele” criterion. b Mutation types of Droplet-CirSeq. The error rates of most of the mutation types were lower than 3.00 X 10 -6 , but the error rates for the transitions C= > T and G= > A were almost one order of magnitude higher than the other types, and the other two transitions, A= > G and T= > C, also showed high error rates. The mutation pattern of the “2X allele” showed the same pattern as with the “1X allele”

    Journal: BMC Genomics

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

    doi: 10.1186/s12864-016-2480-1

    Figure Lengend Snippet: Error rate and mutation types of Droplet-CirSeq. a Error rate of Droplet-CirSeq and Cir-seq. The “1X allele” represents the bases that were supported at least by one circularized DNA, while the “2X allele” represents the bases that were supported by at least two different circularized DNAs. The error rate of Droplet-CirSeq was 5.23 X 10 -5 (±1.54 X 10 -5 ) at the “1X allele” criterion and 3.71 X 10 -6 (±2.34 X 10 -7 ) at the “2X allele” criterion. b Mutation types of Droplet-CirSeq. The error rates of most of the mutation types were lower than 3.00 X 10 -6 , but the error rates for the transitions C= > T and G= > A were almost one order of magnitude higher than the other types, and the other two transitions, A= > G and T= > C, also showed high error rates. The mutation pattern of the “2X allele” showed the same pattern as with the “1X allele”

    Article Snippet: Droplet-CirSeq library preparation Genomic DNA (1 ~ 3 μg) was sheared into 100 ~ 200 bp fragments in Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA) using Covaris S220 in 100 μl volume (shearing condition: duty cycle: 10 %, intensity: 5, cycles per burst: 100, time: 600 s), then purified with the 1.8X Ampure XP beads.

    Techniques: Mutagenesis

    Overview of Droplet-CirSeq. a Droplet-CirSeq workflow. Genomic DNA was sheared into fragments shorter than half the length of the sequencing read and then denatured into single-stranded DNA molecules that were circularized using single-strand DNA ligase. After eliminating the linear DNA using DNA exonucleases, the circularized single-stranded DNA was used for RCA (rolling circle replication). The circularized DNA and RCA reaction mix was added to a RainDrop Source chip to produce water-in-oil emulsion droplets. Generally, approximately 5 ~ 10 million droplets formed in approximately an hour in a 50 μl volume. The droplets containing RCA mix were allowed to continue to react for 4 ~ 16 h in order to amplify enough DNA for standard NGS library preparation in the following steps. Please note that the insert size of the standard NGS libraries must be larger than twice the length of the original circularized DNA to avoid sequencing the same DNA copy twice instead of sequencing two independent-amplified copies. b Error correction. Here is an example to explain the error correction strategy. Multiple copies of the original circularized DNA were examined in every PE read. “A” (red color) represents the base, which may have errors generated during PCR or sequencing. There will be three cases present in the sequencing result: AA (case 1), no errors; AB (case 2), one read error; and BB (case 3), two read errors. B stands for T/C /G. In the following bioinformatics analysis, Case 2 and Case 3 will be filtered except when BB has the same bases, such as TT, GG, or CC (false positive)

    Journal: BMC Genomics

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

    doi: 10.1186/s12864-016-2480-1

    Figure Lengend Snippet: Overview of Droplet-CirSeq. a Droplet-CirSeq workflow. Genomic DNA was sheared into fragments shorter than half the length of the sequencing read and then denatured into single-stranded DNA molecules that were circularized using single-strand DNA ligase. After eliminating the linear DNA using DNA exonucleases, the circularized single-stranded DNA was used for RCA (rolling circle replication). The circularized DNA and RCA reaction mix was added to a RainDrop Source chip to produce water-in-oil emulsion droplets. Generally, approximately 5 ~ 10 million droplets formed in approximately an hour in a 50 μl volume. The droplets containing RCA mix were allowed to continue to react for 4 ~ 16 h in order to amplify enough DNA for standard NGS library preparation in the following steps. Please note that the insert size of the standard NGS libraries must be larger than twice the length of the original circularized DNA to avoid sequencing the same DNA copy twice instead of sequencing two independent-amplified copies. b Error correction. Here is an example to explain the error correction strategy. Multiple copies of the original circularized DNA were examined in every PE read. “A” (red color) represents the base, which may have errors generated during PCR or sequencing. There will be three cases present in the sequencing result: AA (case 1), no errors; AB (case 2), one read error; and BB (case 3), two read errors. B stands for T/C /G. In the following bioinformatics analysis, Case 2 and Case 3 will be filtered except when BB has the same bases, such as TT, GG, or CC (false positive)

    Article Snippet: Droplet-CirSeq library preparation Genomic DNA (1 ~ 3 μg) was sheared into 100 ~ 200 bp fragments in Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA) using Covaris S220 in 100 μl volume (shearing condition: duty cycle: 10 %, intensity: 5, cycles per burst: 100, time: 600 s), then purified with the 1.8X Ampure XP beads.

    Techniques: Sequencing, Chromatin Immunoprecipitation, Next-Generation Sequencing, Amplification, Generated, Polymerase Chain Reaction

    a FN site distribution of the Droplet-CirSeq and Cir-seq. The SEQ_Bias sites are FN sites with a depth less than or equal to 30X. The STR_Bias sites are FN sites with a depth greater than 30X but were still not detected as SNPs due to DNA strand amplification bias. The 3 pg input Droplet-CirSeq method had a lower SEQ_Bias, indicating that it improved the amplification of the poorly amplified region. The 300 pg input Droplet-CirSeq method had a lower STR_Bias, indicating that greater input improved STR_Bias. b Mutation type of FP sites. c True positive SNP frequency distribution for Droplet-CirSeq and Cir-seq; the box width indicates the detected SNP number, the outliers were excluded. d Mutation frequency of FP sites. e FPR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern. f FNR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern

    Journal: BMC Genomics

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

    doi: 10.1186/s12864-016-2480-1

    Figure Lengend Snippet: a FN site distribution of the Droplet-CirSeq and Cir-seq. The SEQ_Bias sites are FN sites with a depth less than or equal to 30X. The STR_Bias sites are FN sites with a depth greater than 30X but were still not detected as SNPs due to DNA strand amplification bias. The 3 pg input Droplet-CirSeq method had a lower SEQ_Bias, indicating that it improved the amplification of the poorly amplified region. The 300 pg input Droplet-CirSeq method had a lower STR_Bias, indicating that greater input improved STR_Bias. b Mutation type of FP sites. c True positive SNP frequency distribution for Droplet-CirSeq and Cir-seq; the box width indicates the detected SNP number, the outliers were excluded. d Mutation frequency of FP sites. e FPR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern. f FNR of different input Droplet-CirSeq libraries after filtering with the mutation frequency pattern

    Article Snippet: Droplet-CirSeq library preparation Genomic DNA (1 ~ 3 μg) was sheared into 100 ~ 200 bp fragments in Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA) using Covaris S220 in 100 μl volume (shearing condition: duty cycle: 10 %, intensity: 5, cycles per burst: 100, time: 600 s), then purified with the 1.8X Ampure XP beads.

    Techniques: Amplification, Mutagenesis

    Proposed role of 2-oxoglutarate (2OG) during cyanophage infection. A. In uninfected cyanobacteria, nitrogen limitation causes 2OG to accumulate, leading to 2OG-dependent binding of NtcA to promoters of nitrogen-stress genes, resulting in their expression. B. Phage infection draws down cellular nitrogen causing N-stress and likely leading to 2OG accumulation. Several cyanophage-encoded enzymes (in bold) suggest that increased 2OG may facilitate phage infection. First, a putative phytanoyl-CoA dioxygenase may convert 2OG to succinate, a major electron donor to respiratory electron transport in cyanobacteria ( Cooley and Vermaas, 2001 ) thus potentially generating energy for the infection process. Second, 2OG-dependent dioxygenase [2OG-Fe(II)] superfamily proteins may function in cyanophage DNA repair ( Weigele et al ., 2007 ). Third, cyanophage genomes have multiple NtcA promoters driving genes encoding diverse functions – possibly exploiting the host NtcA-driven N-stress response system.

    Journal: Environmental Microbiology

    Article Title: Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments

    doi: 10.1111/j.1462-2920.2010.02280.x

    Figure Lengend Snippet: Proposed role of 2-oxoglutarate (2OG) during cyanophage infection. A. In uninfected cyanobacteria, nitrogen limitation causes 2OG to accumulate, leading to 2OG-dependent binding of NtcA to promoters of nitrogen-stress genes, resulting in their expression. B. Phage infection draws down cellular nitrogen causing N-stress and likely leading to 2OG accumulation. Several cyanophage-encoded enzymes (in bold) suggest that increased 2OG may facilitate phage infection. First, a putative phytanoyl-CoA dioxygenase may convert 2OG to succinate, a major electron donor to respiratory electron transport in cyanobacteria ( Cooley and Vermaas, 2001 ) thus potentially generating energy for the infection process. Second, 2OG-dependent dioxygenase [2OG-Fe(II)] superfamily proteins may function in cyanophage DNA repair ( Weigele et al ., 2007 ). Third, cyanophage genomes have multiple NtcA promoters driving genes encoding diverse functions – possibly exploiting the host NtcA-driven N-stress response system.

    Article Snippet: Briefly, 100 µl of cyanophage genomic DNA (1 ng to 2.2 µg) was sheared using Covaris AFA technology and the following conditions: time = 240 s, duty cycle = 5, intensity = 5; cycles per burst = 200 and temperature = 3°C.

    Techniques: Infection, Binding Assay, Expressing