Structured Review

Illumina Inc 100 bp single end sequencing
QTL map. QTL analysis using the binary interval mapping model. Results indicate that the pupal color phenotype is tightly linked to loci on the autosome chromosome 3. A permutation test performed with <t>100,000</t> permutations identified loci showing significant linkage to the phenotype ( P
100 Bp Single End Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "A Chromosome-Scale Assembly of the Bactrocera cucurbitae Genome Provides Insight to the Genetic Basis of white pupae"

Article Title: A Chromosome-Scale Assembly of the Bactrocera cucurbitae Genome Provides Insight to the Genetic Basis of white pupae

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.117.040170

QTL map. QTL analysis using the binary interval mapping model. Results indicate that the pupal color phenotype is tightly linked to loci on the autosome chromosome 3. A permutation test performed with 100,000 permutations identified loci showing significant linkage to the phenotype ( P
Figure Legend Snippet: QTL map. QTL analysis using the binary interval mapping model. Results indicate that the pupal color phenotype is tightly linked to loci on the autosome chromosome 3. A permutation test performed with 100,000 permutations identified loci showing significant linkage to the phenotype ( P

Techniques Used:

2) Product Images from "CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries"

Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries

Journal: Genome Biology

doi: 10.1186/s13059-016-0915-2

A pooled screen for functional validation of CLD. a The screening strategy in SW480 cells. In brief, a pool of mutant SW480 cells harboring 12,471 sgRNA designs against 408 genes was generated by lentiviral infection and antibiotic selection. Fourteen million cells per condition were then treated with PBS (control) or recombinant TRAIL (treatment) for a total of 12 days. Subsequently, the genomic DNA of the samples was extracted and sgRNA composition analyzed by next-generation sequencing ( NGS ). b Comparison of sgRNA sequence counts between two biological replicates demonstrates high reproducibility (Pearson correlation coefficient ~0.79). c Distribution of sgRNAs targeting positive pathway regulators ( CASP8 , CASP3 , FADD , BAX , BID , TNFRSF10A , TNFRSF10B ) in red , negative regulators ( XIAP , BCL2L1 ) in blue , and random, non-targeting sgRNAs in orange between TRAIL ( y-axis ) and PBS ( x-axis ) treated cell populations. d Scatter plot showing relative enrichment of genes ( y-axis ) with their corresponding p value ( x-axis ). Positive regulators are plotted in red , negative regulators in blue , and random, non-targeting sgRNAs in orange. P values were calculated by Wilcoxon rank sum test between 30 sgRNAs of one gene and 200 random, non-targeting sgRNAs. Log 2 fold change is calculated as median log 2 ratio between normalized sgRNA count of TRAIL- over PBS-treated populations. The vertical line marks a p value of 0.05. e – g Median normalized fold change of all sgRNAs targeting three essential TRAIL pathway components. A total of 100 sgRNAs are depicted for each gene. Enriched sgRNAs are colored in red , depleted sgRNAs in grey . The dashed line represents the median fold change of all sgRNAs of the corresponding gene
Figure Legend Snippet: A pooled screen for functional validation of CLD. a The screening strategy in SW480 cells. In brief, a pool of mutant SW480 cells harboring 12,471 sgRNA designs against 408 genes was generated by lentiviral infection and antibiotic selection. Fourteen million cells per condition were then treated with PBS (control) or recombinant TRAIL (treatment) for a total of 12 days. Subsequently, the genomic DNA of the samples was extracted and sgRNA composition analyzed by next-generation sequencing ( NGS ). b Comparison of sgRNA sequence counts between two biological replicates demonstrates high reproducibility (Pearson correlation coefficient ~0.79). c Distribution of sgRNAs targeting positive pathway regulators ( CASP8 , CASP3 , FADD , BAX , BID , TNFRSF10A , TNFRSF10B ) in red , negative regulators ( XIAP , BCL2L1 ) in blue , and random, non-targeting sgRNAs in orange between TRAIL ( y-axis ) and PBS ( x-axis ) treated cell populations. d Scatter plot showing relative enrichment of genes ( y-axis ) with their corresponding p value ( x-axis ). Positive regulators are plotted in red , negative regulators in blue , and random, non-targeting sgRNAs in orange. P values were calculated by Wilcoxon rank sum test between 30 sgRNAs of one gene and 200 random, non-targeting sgRNAs. Log 2 fold change is calculated as median log 2 ratio between normalized sgRNA count of TRAIL- over PBS-treated populations. The vertical line marks a p value of 0.05. e – g Median normalized fold change of all sgRNAs targeting three essential TRAIL pathway components. A total of 100 sgRNAs are depicted for each gene. Enriched sgRNAs are colored in red , depleted sgRNAs in grey . The dashed line represents the median fold change of all sgRNAs of the corresponding gene

Techniques Used: Functional Assay, Mutagenesis, Generated, Infection, Selection, Recombinant, Next-Generation Sequencing, Sequencing

Related Articles

Sequencing:

Article Title: TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
Article Snippet: .. Samples were then pooled in groups of six and subjected to 100-bp single-end sequencing on an Illumina HiSeq instrument ( ). .. Sequence processing and differential expression analysis All sequences were first filtered to remove reads that did not pass Illumina’s chastity filter.

Article Title: Transcriptomic and epigenetic responses to short-term nutrient-exercise stress in humans
Article Snippet: .. Libraries were subjected to 100-bp single-end sequencing on the HiSeq 2500 (Illumina) at the Danish National High-Throughput DNA Sequencing Centre. .. Approximately 8.5 million reads/sample were assigned to genes with 23,373 genes surviving the expression threshold.

Article Title: Syntrophic Growth of Desulfovibrio alaskensis Requires Genes for H2 and Formate Metabolism as Well as Those for Flagellum and Biofilm Formation
Article Snippet: .. RNA was shipped on dry ice to the University of Georgia Genomics facility for 100-bp single-end sequencing with Illumina HiSeq. ..

Article Title: A Chromosome-Scale Assembly of the Bactrocera cucurbitae Genome Provides Insight to the Genetic Basis of white pupae
Article Snippet: .. The ddRAD libraries were each subjected to 100-bp single-end sequencing on a lane of an Illumina HiSequation 2500 Sequencer run in Rapid-Run mode. ..

Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
Article Snippet: .. The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM. ..

Article Title: Exercise training alters the genomic response to acute exercise in human adipose tissue
Article Snippet: .. Last, libraries were quality-controlled using a Bioanalyzer instrument (Agilent Technologies) and subjected to 100-bp single-end sequencing on a HiSeq 2500 system (Illumina) at the Danish National High-Throughput DNA Sequencing Center. .. Pre- and post-training DNA samples from 12 and nine participants (nine paired samples), respectively, were randomly chosen for DNA CpG methylation analysis.

Article Title: DNA demethylation by 5-aza-2′-deoxycytidine is imprinted, targeted to euchromatin, and has limited transcriptional consequences
Article Snippet: .. Indexed libraries were multiplexed for 100-bp single-end sequencing on the Illumina HiSeq 2500 platform at the institutional Epigenomics Shared Facility. .. Given the concordance of extremely limited effects on transcription for different time points and drug dosages, and following recommendations of ENCODE for RNA-seq experiments [ ], we allowed the four treatment conditions to act as biological replicates, increasing confidence in our findings of minimal transcriptional effects of 5-aza-CdR.

Article Title: Learning-dependent chromatin remodeling highlights noncoding regulatory regions linked to autism
Article Snippet: .. Sequencing libraries were quantified using the KAPA Library Quantification Kit, normalized to 10 nM, and submitted to the Penn Genome Frontiers Institute sequencing core at the University of Pennsylvania for 100-bp single-end sequencing on an Illumina HiSeq2000. .. The average sequence depth was 148 million reads, four independent biological replicates per condition.

Sensitive Assay:

Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
Article Snippet: .. The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM. ..

Purification:

Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
Article Snippet: .. The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM. ..

Concentration Assay:

Article Title: CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries
Article Snippet: .. The purified libraries were controlled for correct size using DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM. ..

DNA Sequencing:

Article Title: Transcriptomic and epigenetic responses to short-term nutrient-exercise stress in humans
Article Snippet: .. Libraries were subjected to 100-bp single-end sequencing on the HiSeq 2500 (Illumina) at the Danish National High-Throughput DNA Sequencing Centre. .. Approximately 8.5 million reads/sample were assigned to genes with 23,373 genes surviving the expression threshold.

Article Title: Exercise training alters the genomic response to acute exercise in human adipose tissue
Article Snippet: .. Last, libraries were quality-controlled using a Bioanalyzer instrument (Agilent Technologies) and subjected to 100-bp single-end sequencing on a HiSeq 2500 system (Illumina) at the Danish National High-Throughput DNA Sequencing Center. .. Pre- and post-training DNA samples from 12 and nine participants (nine paired samples), respectively, were randomly chosen for DNA CpG methylation analysis.

High Throughput Screening Assay:

Article Title: Transcriptomic and epigenetic responses to short-term nutrient-exercise stress in humans
Article Snippet: .. Libraries were subjected to 100-bp single-end sequencing on the HiSeq 2500 (Illumina) at the Danish National High-Throughput DNA Sequencing Centre. .. Approximately 8.5 million reads/sample were assigned to genes with 23,373 genes surviving the expression threshold.

Article Title: Exercise training alters the genomic response to acute exercise in human adipose tissue
Article Snippet: .. Last, libraries were quality-controlled using a Bioanalyzer instrument (Agilent Technologies) and subjected to 100-bp single-end sequencing on a HiSeq 2500 system (Illumina) at the Danish National High-Throughput DNA Sequencing Center. .. Pre- and post-training DNA samples from 12 and nine participants (nine paired samples), respectively, were randomly chosen for DNA CpG methylation analysis.

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    Illumina Inc 100 bp single end
    Ancestry results for samples from the southeastern portion of the range. Ancestry proportions were determined using Structure with 50,000 and <t>100,000</t> burn‐in and MCMC iterations and the previously determined optimal K ‐value of 2
    100 Bp Single End, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/100 bp single end/product/Illumina Inc
    Average 90 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
    100 bp single end - by Bioz Stars, 2020-07
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    91
    Illumina Inc 100 bp paired single end
    Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, <t>100</t> µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P
    100 Bp Paired Single End, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/100 bp paired single end/product/Illumina Inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    100 bp paired single end - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    Ancestry results for samples from the southeastern portion of the range. Ancestry proportions were determined using Structure with 50,000 and 100,000 burn‐in and MCMC iterations and the previously determined optimal K ‐value of 2

    Journal: Evolutionary Applications

    Article Title: Population assignment and local adaptation along an isolation‐by‐distance gradient in Pacific cod (Gadus macrocephalus). Population assignment and local adaptation along an isolation‐by‐distance gradient in Pacific cod (Gadus macrocephalus)

    doi: 10.1111/eva.12639

    Figure Lengend Snippet: Ancestry results for samples from the southeastern portion of the range. Ancestry proportions were determined using Structure with 50,000 and 100,000 burn‐in and MCMC iterations and the previously determined optimal K ‐value of 2

    Article Snippet: Libraries were pooled within samples in 10 nM concentrations and sequenced in 100‐bp single‐end reads on a HiSeq2000 (Illumina, Inc., San Diego, CA) at the University of Oregon Genomics and Cell Characterization Core Facility (GC3F, Eugene, Oregon).

    Techniques:

    LD decay (mean r 2 ) estimated in three populations (as defined by population genetic structure) using 3321 desi (A) and 8592 kabuli (B) SNPs, respectively . For LD decay, the r 2 -value of the marker physical distance of 0 kb is considered 1. The plotted curved lines indicate the mean r 2 -values among markers spaced with uniform 100 kb physical intervals from 0 to 1000 kb. The plotted line in uppermost panel I indicate the mean r 2 -values among markers spaced with uniform 10 kb physical intervals from 0 to 100 kb. The marked line in panels I, II indicate the mean r 2 -values among markers spaced with uniform 10 and 20 kb physical intervals from 0 to 100 kb and 0 to 200 kb, respectively. “All” includes the LD decay across entire three populations.

    Journal: Frontiers in Plant Science

    Article Title: Employing genome-wide SNP discovery and genotyping strategy to extrapolate the natural allelic diversity and domestication patterns in chickpea

    doi: 10.3389/fpls.2015.00162

    Figure Lengend Snippet: LD decay (mean r 2 ) estimated in three populations (as defined by population genetic structure) using 3321 desi (A) and 8592 kabuli (B) SNPs, respectively . For LD decay, the r 2 -value of the marker physical distance of 0 kb is considered 1. The plotted curved lines indicate the mean r 2 -values among markers spaced with uniform 100 kb physical intervals from 0 to 1000 kb. The plotted line in uppermost panel I indicate the mean r 2 -values among markers spaced with uniform 10 kb physical intervals from 0 to 100 kb. The marked line in panels I, II indicate the mean r 2 -values among markers spaced with uniform 10 and 20 kb physical intervals from 0 to 100 kb and 0 to 200 kb, respectively. “All” includes the LD decay across entire three populations.

    Article Snippet: These libraries were pooled together (following Elshire et al., ; Spindel et al., ) and sequenced (100-bp single end) using Illumina HiSeq 2000.

    Techniques: Marker

    A genome scan plot depicting the density of SNPs physically mapped across eight chromosomes of desi (A) and kabuli (B) genomes at 100-kb sliding window . The SNP density is represented by the number of SNPs mapped within 1-Mb physical intervals across chromosomes.

    Journal: Frontiers in Plant Science

    Article Title: Employing genome-wide SNP discovery and genotyping strategy to extrapolate the natural allelic diversity and domestication patterns in chickpea

    doi: 10.3389/fpls.2015.00162

    Figure Lengend Snippet: A genome scan plot depicting the density of SNPs physically mapped across eight chromosomes of desi (A) and kabuli (B) genomes at 100-kb sliding window . The SNP density is represented by the number of SNPs mapped within 1-Mb physical intervals across chromosomes.

    Article Snippet: These libraries were pooled together (following Elshire et al., ; Spindel et al., ) and sequenced (100-bp single end) using Illumina HiSeq 2000.

    Techniques:

    Gene Correlation Network Analysis (A) Hierarchical cluster dendrogram groups 21,143 genes into distinct co-expression modules identified using weighted gene correlation network analysis. Six major modules are indicated as colored boxes (Mod1, black; Mod2, salmon; Mod3, brown; Mod4, yellow; Mod5, midnight blue; Mod6, blue). (B) Heatmap showing relative expression of genes in the six major modules across ten samples of two sexes. The color values are the standardized Z scores of gene expression. (C) Percentage of genes that are previously known to be enriched in specific cells or tissues across different modules. Previously known gene lists include for panneural enriched ( Spencer et al., 2011 ), sperm and oocyte enriched ( Reinke et al., 2004 ), and cuticle collagen enriched ( Page and Johnstone, 2007 ). Number of genes was normalized in order to avoid the effect of different module sizes (y axis). (D and E) Tissue-specific expression of reporter transgenes for two C-type lectin domain ( clec ) genes chosen from Mod1 (D) and Mod3 (E) in both sexes. Arrow indicates expression in head neurons in (D) or in vas deferens of male in (E). The magnified view of the arrowed region is shown at the bottom of each image. In all images, anterior is left and posterior is right. The scale bars represent 100 μm.

    Journal: Cell reports

    Article Title: Gene Function Prediction Based on Developmental Transcriptomes of the Two Sexes in C. elegans

    doi: 10.1016/j.celrep.2016.09.051

    Figure Lengend Snippet: Gene Correlation Network Analysis (A) Hierarchical cluster dendrogram groups 21,143 genes into distinct co-expression modules identified using weighted gene correlation network analysis. Six major modules are indicated as colored boxes (Mod1, black; Mod2, salmon; Mod3, brown; Mod4, yellow; Mod5, midnight blue; Mod6, blue). (B) Heatmap showing relative expression of genes in the six major modules across ten samples of two sexes. The color values are the standardized Z scores of gene expression. (C) Percentage of genes that are previously known to be enriched in specific cells or tissues across different modules. Previously known gene lists include for panneural enriched ( Spencer et al., 2011 ), sperm and oocyte enriched ( Reinke et al., 2004 ), and cuticle collagen enriched ( Page and Johnstone, 2007 ). Number of genes was normalized in order to avoid the effect of different module sizes (y axis). (D and E) Tissue-specific expression of reporter transgenes for two C-type lectin domain ( clec ) genes chosen from Mod1 (D) and Mod3 (E) in both sexes. Arrow indicates expression in head neurons in (D) or in vas deferens of male in (E). The magnified view of the arrowed region is shown at the bottom of each image. In all images, anterior is left and posterior is right. The scale bars represent 100 μm.

    Article Snippet: Library preparation followed the protocol described at http://wasp.einstein.yu.edu/index.php/Main_Page , and sequences were determined for 100-bp single-end reads using the Illumina HiSeq2500.

    Techniques: Expressing, Polyacrylamide Gel Electrophoresis

    Identification of Semen Protein Genes (A) Schematic of procedure to identify and validate semen protein genes from the RNA-seq data. (B) The anatomical location of seminal vesicle, valve region, and vas deferens in male gonad (gray). (C) Tissue-specific expression of reporter transgenes for three semen candidate genes in male. Arrows indicate expression in seminal vesicle ( ins-31 ), vas deferens ( F59B2.12 ), and the valve region of vas deferens ( B0207.5 ). Arrowhead indicates signal from co-injection marker ttx-3::GFP . In all images, anterior is left and posterior is right. (D) Protein localization identified using translational reporters for ins-31 and F59B2.12 . The Nomarski images show localization within the vesicular structures in the seminal vesicle region for INS-31 and in vas deferens for F59B2.12 protein. (E) Time course images showing transfer of INS-31 from male to hermaphrodite during mating. To facilitate observation of mating behavior, slower-moving unc-119 mutant hermaphrodites were used. INS-31::sfGFP (green) is visible within the seminal vesicle of a male before spicule insertion (a) and moves into the lumen of vas deferens after insertion until ejaculation (b). During ejaculation, INS-31::sfGFP is transferred into the vulva region of the hermaphrodite (c) and remains diffused in the uterus after mating (d). (F) A Venn diagram showing the comparison of semen candidate gene lists obtained from this study, SPELL, and WormNet. The numbers of candidate genes and genes belonging to Mod3 (parentheses) are indicated. The scale bars represent 100 μm in (C) and 20 μm in (D).

    Journal: Cell reports

    Article Title: Gene Function Prediction Based on Developmental Transcriptomes of the Two Sexes in C. elegans

    doi: 10.1016/j.celrep.2016.09.051

    Figure Lengend Snippet: Identification of Semen Protein Genes (A) Schematic of procedure to identify and validate semen protein genes from the RNA-seq data. (B) The anatomical location of seminal vesicle, valve region, and vas deferens in male gonad (gray). (C) Tissue-specific expression of reporter transgenes for three semen candidate genes in male. Arrows indicate expression in seminal vesicle ( ins-31 ), vas deferens ( F59B2.12 ), and the valve region of vas deferens ( B0207.5 ). Arrowhead indicates signal from co-injection marker ttx-3::GFP . In all images, anterior is left and posterior is right. (D) Protein localization identified using translational reporters for ins-31 and F59B2.12 . The Nomarski images show localization within the vesicular structures in the seminal vesicle region for INS-31 and in vas deferens for F59B2.12 protein. (E) Time course images showing transfer of INS-31 from male to hermaphrodite during mating. To facilitate observation of mating behavior, slower-moving unc-119 mutant hermaphrodites were used. INS-31::sfGFP (green) is visible within the seminal vesicle of a male before spicule insertion (a) and moves into the lumen of vas deferens after insertion until ejaculation (b). During ejaculation, INS-31::sfGFP is transferred into the vulva region of the hermaphrodite (c) and remains diffused in the uterus after mating (d). (F) A Venn diagram showing the comparison of semen candidate gene lists obtained from this study, SPELL, and WormNet. The numbers of candidate genes and genes belonging to Mod3 (parentheses) are indicated. The scale bars represent 100 μm in (C) and 20 μm in (D).

    Article Snippet: Library preparation followed the protocol described at http://wasp.einstein.yu.edu/index.php/Main_Page , and sequences were determined for 100-bp single-end reads using the Illumina HiSeq2500.

    Techniques: RNA Sequencing Assay, Expressing, Injection, Marker, Mutagenesis

    Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Staining, Transduction, shRNA, Quantitative RT-PCR

    TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Quantitative RT-PCR, Isolation, Mouse Assay, Staining, Transduction, Western Blot, Expressing, Blocking Assay, Activation Assay

    TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Mouse Assay, Injection, Staining, Marker

    Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Staining, Western Blot, Confocal Microscopy, Isolation, Marker, Expressing, Mouse Assay

    Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Expressing, Genome Wide, RNA Sequencing Assay, Labeling, Quantitative RT-PCR, Isolation, Confocal Microscopy, Staining, Marker, Mouse Assay