hdgfrp2 cryptic exon  (New England Biolabs)


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    New England Biolabs hdgfrp2 cryptic exon
    Monarch Plasmid Miniprep Kit
    Monarch Plasmid Miniprep Kit 250 preps
    https://www.bioz.com/result/hdgfrp2 cryptic exon/product/New England Biolabs
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    hdgfrp2 cryptic exon - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43"

    Article Title: Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-017-1701-2

    Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.
    Figure Legend Snippet: Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    2) Product Images from "TOR coordinates nucleotide availability with ribosome biogenesis in plants"

    Article Title: TOR coordinates nucleotide availability with ribosome biogenesis in plants

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.927418

    Cytosolic PRS (PRS4) drives plant development and TOR activity in N. benthamiana . (A) Silencing PRS4 drastically reduces TOR activity. S6K-pT449 levels reflect TOR activity, because S6K-T449 is a direct substrate of TOR ( Xiong and Sheen, 2012 ). S6K-pT449 and total S6K levels were assayed by Western blots in knockdown TRV:: NbPRS4 plants or mock controls (representative images shown here). Quantification of band densities confirmed that S6K-pT449 levels decrease ∼5-fold in TRV:: NbPRS4 knockdowns, but total S6K levels are not affected in TRV:: NbPRS4 . (B) PRS4 is required for shoot development. There are fewer leaves in TRV:: NbPRS4 knockdowns, and the leaves are misshapen and small. We observed individual-to-individual variation in phenotypic severity after silencing PRS4 by VIGS; a representative of the “moderate” TRV:: NbPRS4 phenotype and of the “severe” TRV:: NbPRS4 phenotype are shown. Outlines of leaf shapes are shown, including all leaves with silenced PRS4 expression (i.e., only leaves above the primary infected leaf), with the oldest leaf on the left and the youngest leaf on the right. (C) Silencing PRS4 impairs cell expansion and cell division. Epidermal pavement cell shape was not dramatically altered in TRV:: NbPRS4 knockdowns, but epidermal pavement cell size was significantly lower. This difference in cell size is insufficient to account for the decrease in total leaf area shown in panel B; therefore, there are also fewer epidermal pavement cells in TRV:: NbPRS4 . (D) We did not observe clear effects of silencing PRS4 on vegetative shoot apical meristem morphology.
    Figure Legend Snippet: Cytosolic PRS (PRS4) drives plant development and TOR activity in N. benthamiana . (A) Silencing PRS4 drastically reduces TOR activity. S6K-pT449 levels reflect TOR activity, because S6K-T449 is a direct substrate of TOR ( Xiong and Sheen, 2012 ). S6K-pT449 and total S6K levels were assayed by Western blots in knockdown TRV:: NbPRS4 plants or mock controls (representative images shown here). Quantification of band densities confirmed that S6K-pT449 levels decrease ∼5-fold in TRV:: NbPRS4 knockdowns, but total S6K levels are not affected in TRV:: NbPRS4 . (B) PRS4 is required for shoot development. There are fewer leaves in TRV:: NbPRS4 knockdowns, and the leaves are misshapen and small. We observed individual-to-individual variation in phenotypic severity after silencing PRS4 by VIGS; a representative of the “moderate” TRV:: NbPRS4 phenotype and of the “severe” TRV:: NbPRS4 phenotype are shown. Outlines of leaf shapes are shown, including all leaves with silenced PRS4 expression (i.e., only leaves above the primary infected leaf), with the oldest leaf on the left and the youngest leaf on the right. (C) Silencing PRS4 impairs cell expansion and cell division. Epidermal pavement cell shape was not dramatically altered in TRV:: NbPRS4 knockdowns, but epidermal pavement cell size was significantly lower. This difference in cell size is insufficient to account for the decrease in total leaf area shown in panel B; therefore, there are also fewer epidermal pavement cells in TRV:: NbPRS4 . (D) We did not observe clear effects of silencing PRS4 on vegetative shoot apical meristem morphology.

    Techniques Used: Activity Assay, Western Blot, Expressing, Infection

    Silencing key genes in nucleotide biosynthesis inhibits TOR activity. (A) Nucleotide biosynthesis is necessary for normal shoot development and physiology. Silencing genes downstream of PRS4 in nucleotide biosynthesis in N. benthamiana reduced leaf number and size, disrupted leaf shape, and caused chlorosis, similar to the phenotypes observed in TRV:: NbPRS4 plants. Each gene was silenced in at least six plants per experiment, and the entire experiment was replicated three times; representative individuals of each silenced gene are shown. (B) Silencing nucleotide biosynthesis genes lowers TOR activity. S6K-pT449 levels are strongly reduced in silenced plants compared to mock-infected controls, and the S6K-pT449/S6K ratios are consistently lower.
    Figure Legend Snippet: Silencing key genes in nucleotide biosynthesis inhibits TOR activity. (A) Nucleotide biosynthesis is necessary for normal shoot development and physiology. Silencing genes downstream of PRS4 in nucleotide biosynthesis in N. benthamiana reduced leaf number and size, disrupted leaf shape, and caused chlorosis, similar to the phenotypes observed in TRV:: NbPRS4 plants. Each gene was silenced in at least six plants per experiment, and the entire experiment was replicated three times; representative individuals of each silenced gene are shown. (B) Silencing nucleotide biosynthesis genes lowers TOR activity. S6K-pT449 levels are strongly reduced in silenced plants compared to mock-infected controls, and the S6K-pT449/S6K ratios are consistently lower.

    Techniques Used: Activity Assay, Infection

    Silencing PRS4 reprograms the transcriptome to repress ribosome biogenesis. (A) Scatterplots of gene expression changes in N. benthamiana after VIGS. 4,986 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe phenotypes and mock plants (top panel), but only 489 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe versus moderate phenotypes (middle panel). Principal component analysis demonstrates that the mock-treated transcriptomes are readily distinguished from the TRV:: NbPRS4 knockdowns, but that the TRV:: NbPRS4 transcriptomes from plants with severe or moderate phenotypes are not grouped separately. (B) MapMan functional analysis of DEGs in TRV:: NbPRS4 revealed 48 significantly-affected categories ( p adj .
    Figure Legend Snippet: Silencing PRS4 reprograms the transcriptome to repress ribosome biogenesis. (A) Scatterplots of gene expression changes in N. benthamiana after VIGS. 4,986 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe phenotypes and mock plants (top panel), but only 489 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe versus moderate phenotypes (middle panel). Principal component analysis demonstrates that the mock-treated transcriptomes are readily distinguished from the TRV:: NbPRS4 knockdowns, but that the TRV:: NbPRS4 transcriptomes from plants with severe or moderate phenotypes are not grouped separately. (B) MapMan functional analysis of DEGs in TRV:: NbPRS4 revealed 48 significantly-affected categories ( p adj .

    Techniques Used: Expressing, Functional Assay

    3) Product Images from "The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein"

    Article Title: The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879437

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P

    Techniques Used: Expressing, Infection, In Vitro, Amplification, Real-time Polymerase Chain Reaction

    The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vivo, Infection, Quantitative RT-PCR, Titration, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Amplification, Infection, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Infection, Real-time Polymerase Chain Reaction, Expressing, Amplification, Two Tailed Test

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: Expressing, Infection, In Vivo, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

    4) Product Images from "CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti"

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58731-y

    RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p
    Figure Legend Snippet: RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p

    Techniques Used: Activity Assay, Injection, Functional Assay, One-tailed Test

    5) Product Images from "Staphylococcus aureus Cas9 is a multiple-turnover enzyme"

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme

    Journal: RNA

    doi: 10.1261/rna.067355.118

    S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA
    Figure Legend Snippet: S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA

    Techniques Used:

    6) Product Images from "Evolution of a General RNA-Cleaving FANA Enzyme"

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07611-1

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
    Figure Legend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Techniques Used: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis

    7) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.
    Figure Legend Snippet: Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.

    Techniques Used: Sequencing

    8) Product Images from "lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17"

    Article Title: lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11018

    lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P
    Figure Legend Snippet: lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P
    Figure Legend Snippet: lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P

    Techniques Used: Over Expression, Expressing

    Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used:

    lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P
    Figure Legend Snippet: lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P

    Techniques Used: Over Expression, Transfection, Isolation

    Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used: Expressing

    9) Product Images from "Filter paper-based spin column method for cost-efficient DNA or RNA purification"

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203011

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Figure Legend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Techniques Used: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    10) Product Images from "Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential"

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Figure Legend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    11) Product Images from "Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior"

    Article Title: Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01569

    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Figure Legend Snippet: CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Techniques Used: CRISPR, Introduce, Sequencing, Polymerase Chain Reaction, Amplification

    12) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    13) Product Images from "Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA"

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.01328-19

    RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.
    Figure Legend Snippet: RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.

    Techniques Used: In Vitro, Binding Assay, Agarose Gel Electrophoresis, Protein Binding, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Amplification, Marker

    14) Product Images from "Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals"

    Article Title: Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

    Journal: bioRxiv

    doi: 10.1101/808642

    A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p
    Figure Legend Snippet: A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p

    Techniques Used: RNA Sequencing Assay, Staining, Sequencing, Immunostaining, Two Tailed Test

    15) Product Images from "A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters"

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    Journal: bioRxiv

    doi: 10.1101/834630

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Figure Legend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Techniques Used: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    16) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    17) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

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

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    18) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    19) Product Images from "Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing"

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

    Journal: Microbial Cell

    doi: 10.15698/mic2018.09.645

    FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.
    Figure Legend Snippet: FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software

    20) Product Images from "Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection"

    Article Title: Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection

    Journal: Viruses

    doi: 10.3390/v10090499

    Primer walking for elucidation of ORF-A 3′ terminus. Strand-specific RT-PCR of BoHV-1.1-infected cell mRNA extracts was was performed on cDNA produced from BoHV-1 infected cells using 16 different reverse primers that anneal further down (3′) to the viral genome. Successful amplification allowed for capture of increasing lengths of the ORF-A transcript sequence. RT indicates the presence (+) or absence (-) of a retrotranscriptase step with mRNA as a template. ( a ) Amplicons were produced with primers 1–8 using a 1-min extension time during PCR. ( b ) Amplicons were produced using primers 9-16 with a 2-min extension time to account for the increase in amplicon length.
    Figure Legend Snippet: Primer walking for elucidation of ORF-A 3′ terminus. Strand-specific RT-PCR of BoHV-1.1-infected cell mRNA extracts was was performed on cDNA produced from BoHV-1 infected cells using 16 different reverse primers that anneal further down (3′) to the viral genome. Successful amplification allowed for capture of increasing lengths of the ORF-A transcript sequence. RT indicates the presence (+) or absence (-) of a retrotranscriptase step with mRNA as a template. ( a ) Amplicons were produced with primers 1–8 using a 1-min extension time during PCR. ( b ) Amplicons were produced using primers 9-16 with a 2-min extension time to account for the increase in amplicon length.

    Techniques Used: Chromosome Walking, Reverse Transcription Polymerase Chain Reaction, Infection, Produced, Amplification, Sequencing, Polymerase Chain Reaction

    21) Product Images from "Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes"

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00186

    UV–vis absorption and second derivative (D 2 ) spectra (Panel A) and RR spectra in the high frequency region (Panel B) of the coproheme complexes with WT and the Q187A, M149A/Q187A, and M149A mutants. The band wavelengths and wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of the spectra in Panel A is expanded 9-fold. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample of 5 mW, average of 9 spectra with a 90 min integration time (WT), 14 spectra with a 140 min integration time (Q187A), 10 spectra with a 100 min integration time (M149A/Q187A), and 4 spectra with a 40 min integration time (M149A).
    Figure Legend Snippet: UV–vis absorption and second derivative (D 2 ) spectra (Panel A) and RR spectra in the high frequency region (Panel B) of the coproheme complexes with WT and the Q187A, M149A/Q187A, and M149A mutants. The band wavelengths and wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of the spectra in Panel A is expanded 9-fold. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample of 5 mW, average of 9 spectra with a 90 min integration time (WT), 14 spectra with a 140 min integration time (Q187A), 10 spectra with a 100 min integration time (M149A/Q187A), and 4 spectra with a 40 min integration time (M149A).

    Techniques Used:

    UV–vis (panel A) and RR (panel B) spectra in the low (left) and high (right) frequency regions of the 12 CO adducts of the coproheme complexes of Mb, WT, M149A, M149A/Q187A, Q187A, and coproheme. The frequencies of the ν(FeC), δ(FeCO), and ν(CO) modes are indicated in red. The spectra have been shifted along the ordinate axis to allow better visualization. Panel A: the 480–700 nm region is expanded 10-fold. Panel B: experimental conditions: Mb and coproheme: λ exc 406.7 nm, laser power at the sample 5 mW, average of 4 spectra with 40 min integration time and 10 spectra with 100 min integration time in the low and high frequency regions, respectively (Mb), average of 6 spectra with 60 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively (coproheme); WT and its mutants, λ exc 413.1 nm, laser power at the sample 1–3 mW; average of 28 spectra with 280 min integration time and 22 spectra with 220 min integration time in the low and high frequency regions, respectively (WT), average of 6 spectra with 60 min integration time and 18 spectra with 180 min integration time in the low and high frequency regions, respectively (M149A), average of 6 spectra with 60 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (M149A/Q187A), and average of 9 spectra with 90 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (Q187A).
    Figure Legend Snippet: UV–vis (panel A) and RR (panel B) spectra in the low (left) and high (right) frequency regions of the 12 CO adducts of the coproheme complexes of Mb, WT, M149A, M149A/Q187A, Q187A, and coproheme. The frequencies of the ν(FeC), δ(FeCO), and ν(CO) modes are indicated in red. The spectra have been shifted along the ordinate axis to allow better visualization. Panel A: the 480–700 nm region is expanded 10-fold. Panel B: experimental conditions: Mb and coproheme: λ exc 406.7 nm, laser power at the sample 5 mW, average of 4 spectra with 40 min integration time and 10 spectra with 100 min integration time in the low and high frequency regions, respectively (Mb), average of 6 spectra with 60 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively (coproheme); WT and its mutants, λ exc 413.1 nm, laser power at the sample 1–3 mW; average of 28 spectra with 280 min integration time and 22 spectra with 220 min integration time in the low and high frequency regions, respectively (WT), average of 6 spectra with 60 min integration time and 18 spectra with 180 min integration time in the low and high frequency regions, respectively (M149A), average of 6 spectra with 60 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (M149A/Q187A), and average of 9 spectra with 90 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (Q187A).

    Techniques Used:

    22) Product Images from "Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes"

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00186

    UV–vis absorption and second derivative (D 2 ) spectra of coproheme and the coproheme complexes with LmChdC WT, its M149A mutant, and Mb. The band wavelengths assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively, (see text). The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of coproheme and the coproheme complexes spectra is expanded 20- and 9-fold, respectively. The excitation wavelengths used for the RR experiments are also shown in light violet (the 356.4 nm line) and in violet (the 406.7 nm line).
    Figure Legend Snippet: UV–vis absorption and second derivative (D 2 ) spectra of coproheme and the coproheme complexes with LmChdC WT, its M149A mutant, and Mb. The band wavelengths assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively, (see text). The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of coproheme and the coproheme complexes spectra is expanded 20- and 9-fold, respectively. The excitation wavelengths used for the RR experiments are also shown in light violet (the 356.4 nm line) and in violet (the 406.7 nm line).

    Techniques Used: Mutagenesis

    High frequency region RR spectra obtained at room temperature, with the 356.4 (Panel A) and 406.7 nm (Panel B) excitation wavelengths, of coproheme, and the coproheme complexes of LmChdC WT, its M149A mutant, and Mb. The band wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively (see text). The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: (A) laser power at the sample 5 mW, average of 10 spectra with 120 min integration time (Coproheme); laser power at the sample 2 mW; average of 7 spectra with 70 min integration time (WT), 8 spectra with 80 min integration time (M149A), and 24 spectra with 240 min integration time (Mb). (B) laser power at the sample 5 mW; average of 2 spectra with 10 min integration time with 1800 grating (Coproheme), 9 spectra with 90 min integration time (WT), 4 spectra with 40 min integration time (M149A), and 6 spectra with 60 min integration time (Mb).
    Figure Legend Snippet: High frequency region RR spectra obtained at room temperature, with the 356.4 (Panel A) and 406.7 nm (Panel B) excitation wavelengths, of coproheme, and the coproheme complexes of LmChdC WT, its M149A mutant, and Mb. The band wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively (see text). The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: (A) laser power at the sample 5 mW, average of 10 spectra with 120 min integration time (Coproheme); laser power at the sample 2 mW; average of 7 spectra with 70 min integration time (WT), 8 spectra with 80 min integration time (M149A), and 24 spectra with 240 min integration time (Mb). (B) laser power at the sample 5 mW; average of 2 spectra with 10 min integration time with 1800 grating (Coproheme), 9 spectra with 90 min integration time (WT), 4 spectra with 40 min integration time (M149A), and 6 spectra with 60 min integration time (Mb).

    Techniques Used: Mutagenesis

    Cross-linking of heme b to LmChdC mediated by excess hydrogen peroxide. Mass spectrometric analysis of the entire protein of LmChdC WT (apo-form 31 977.2 Da; cross-linked 32 590.5 Da, black) and LmChdC M149A (31 917.0 Da, red). The green line with its label shows the mass difference between apo-LmChd WT and holo-LmChdC WT. The Coproheme-LmChdC complexes were titrated with H 2 O 2 up to a 2-fold excess; subsequently, the mass spectroscopic measurements were performed on heme b -LmChdC WT and heme b -LmChdC M149A.
    Figure Legend Snippet: Cross-linking of heme b to LmChdC mediated by excess hydrogen peroxide. Mass spectrometric analysis of the entire protein of LmChdC WT (apo-form 31 977.2 Da; cross-linked 32 590.5 Da, black) and LmChdC M149A (31 917.0 Da, red). The green line with its label shows the mass difference between apo-LmChd WT and holo-LmChdC WT. The Coproheme-LmChdC complexes were titrated with H 2 O 2 up to a 2-fold excess; subsequently, the mass spectroscopic measurements were performed on heme b -LmChdC WT and heme b -LmChdC M149A.

    Techniques Used:

    Heme coordination for the coproheme complexes of LmChdC, its M149A mutant, and Mb determined by RR and EPR spectroscopy at room and low temperatures..
    Figure Legend Snippet: Heme coordination for the coproheme complexes of LmChdC, its M149A mutant, and Mb determined by RR and EPR spectroscopy at room and low temperatures..

    Techniques Used: Mutagenesis, Electron Paramagnetic Resonance, Spectroscopy

    23) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.
    Figure Legend Snippet: Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.

    Techniques Used: Sequencing

    24) Product Images from "A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins"

    Article Title: A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007533

    MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.
    Figure Legend Snippet: MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.

    Techniques Used: Synthesized, Amplification, Polymerase Chain Reaction, Marker

    25) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.
    Figure Legend Snippet: RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Concentration Assay, Negative Control

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Figure Legend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    26) Product Images from "Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies"

    Article Title: Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies

    Journal: Genes

    doi: 10.3390/genes10020079

    Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.
    Figure Legend Snippet: Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.

    Techniques Used: Functional Assay

    27) Product Images from "Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy"

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41313-y

    Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).
    Figure Legend Snippet: Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

    28) Product Images from "High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments"

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz851

    Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.
    Figure Legend Snippet: Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.

    Techniques Used: Labeling, Construct, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Purification, Ligation

    Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.
    Figure Legend Snippet: Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.

    Techniques Used: Construct, Labeling, Ligation, Plasmid Preparation, Polymerase Chain Reaction, Purification, In Vitro, Amplification

    29) Product Images from "Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice"

    Article Title: Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23830-4

    Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii .
    Figure Legend Snippet: Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii .

    Techniques Used: Polymerase Chain Reaction, Amplification, Infection, Mouse Assay, Mass Spectrometry, Isolation, Cell Culture, Positive Control, Molecular Weight, Marker

    30) Product Images from "Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator"

    Article Title: Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008729

    Generation and validation of Venus::BMAL1 mouse. A) sgRNA targeting design across Bmal1 start codon (ATG in bold, shading indicates gene coding sequence). HDR targeting cassette design, cut site indicated by scissors, corresponding homology arms by red boxes and Venus-linker-linker in yellow. Black arrows show post-HDR allele primers for genotyping. B) WB in mouse lung with anti-BMAL1 antiserum, showing increased molecular weight of Venus::BMAL1 (V/V). C) There was no difference in the pattern of BMAL1 expression (shown by IHC) between Venus and non-Venus mouse hip. “No antibody control” confirms the specificity of the antiserum. D) Top: Representative confocal images of SCN from brain sections taken from Venus::BMAL1 animals. Venus::BMAL1 showed nearly 100% registration with endogenous BMAL1 in the SCN from heterozygous animals. Expression of Venus::BMAL1 (yellow) was revealed by confocal fluorescence imaging and IF with an anti-BMAL1 antibody (red). Bottom: Venus::BMAL1-positive localisation was compared with nuclear staining of DAPI. Venus::BMAL1 was predominantly expressed in the nuclei of cells in the SCN. Scale bars as written in Figure. E) Representative confocal micrographs showing close-up images of cells in fixed SCN sections where Venus::BMAL1 (green) is colocalised with SCN neuropeptides (VIP, AVP and GRP shown in red) by IHC and co-stained with DAPI (blue). Colocalised cells are indicated by arrows shown in orange. Red arrows show gaps in Venus::BMAL1 signal which align with GRP cells. F) Percentage neuropeptide-immunostained cells that co-localise with Venus::BMAL1-positive cells (n = 4 animals). G) Representative Venus::BMAL1 signals in femoral head articular cartilage and intervertebral disc tissues (top) and in primary chondrocyte, fibroblast and IVD cultures (bottom). The IVD preparation was co-stained with DAPI, shown below the fluorescence image. Scale bars as written in Figure. H) Representative visualisation of Venus::BMAL1 fluorescence in MEF and chondrocyte nuclei generated in Imaris from Z-stack spectral imaging. Spectral linear unmixing revealed nuclear localization of Venus::BMAL1, with most of the cytoplasmic fluorescence attributed to auto-fluorescence. Yellow: Venus::BMAL1; Red: CellMask Deep Red Plasma membrane stain; blue: auto-fluorescence.
    Figure Legend Snippet: Generation and validation of Venus::BMAL1 mouse. A) sgRNA targeting design across Bmal1 start codon (ATG in bold, shading indicates gene coding sequence). HDR targeting cassette design, cut site indicated by scissors, corresponding homology arms by red boxes and Venus-linker-linker in yellow. Black arrows show post-HDR allele primers for genotyping. B) WB in mouse lung with anti-BMAL1 antiserum, showing increased molecular weight of Venus::BMAL1 (V/V). C) There was no difference in the pattern of BMAL1 expression (shown by IHC) between Venus and non-Venus mouse hip. “No antibody control” confirms the specificity of the antiserum. D) Top: Representative confocal images of SCN from brain sections taken from Venus::BMAL1 animals. Venus::BMAL1 showed nearly 100% registration with endogenous BMAL1 in the SCN from heterozygous animals. Expression of Venus::BMAL1 (yellow) was revealed by confocal fluorescence imaging and IF with an anti-BMAL1 antibody (red). Bottom: Venus::BMAL1-positive localisation was compared with nuclear staining of DAPI. Venus::BMAL1 was predominantly expressed in the nuclei of cells in the SCN. Scale bars as written in Figure. E) Representative confocal micrographs showing close-up images of cells in fixed SCN sections where Venus::BMAL1 (green) is colocalised with SCN neuropeptides (VIP, AVP and GRP shown in red) by IHC and co-stained with DAPI (blue). Colocalised cells are indicated by arrows shown in orange. Red arrows show gaps in Venus::BMAL1 signal which align with GRP cells. F) Percentage neuropeptide-immunostained cells that co-localise with Venus::BMAL1-positive cells (n = 4 animals). G) Representative Venus::BMAL1 signals in femoral head articular cartilage and intervertebral disc tissues (top) and in primary chondrocyte, fibroblast and IVD cultures (bottom). The IVD preparation was co-stained with DAPI, shown below the fluorescence image. Scale bars as written in Figure. H) Representative visualisation of Venus::BMAL1 fluorescence in MEF and chondrocyte nuclei generated in Imaris from Z-stack spectral imaging. Spectral linear unmixing revealed nuclear localization of Venus::BMAL1, with most of the cytoplasmic fluorescence attributed to auto-fluorescence. Yellow: Venus::BMAL1; Red: CellMask Deep Red Plasma membrane stain; blue: auto-fluorescence.

    Techniques Used: Sequencing, Western Blot, Molecular Weight, Expressing, Immunohistochemistry, Fluorescence, Imaging, Staining, Generated

    31) Product Images from "Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential"

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Figure Legend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    32) Product Images from "Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae"

    Article Title: Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae

    Journal: bioRxiv

    doi: 10.1101/2020.03.28.013177

    Identification and functional analysis of predicted transcription-activator like effectors (TALEs) from endofungal Burkholderia species (BATs). a , Schematic illustration of the gene clusters encoding BAT1 (RBRH_01844), BAT2 (RBRH_01776), and BAT3 (RBRH_01777) indicated in red. b , Mode of action of TALEs from Xanthomonas sp. TALEs are secreted into plant cells via the T3SS, translocate to the nucleus, and induce expression of target genes 22 . c , Schematic representation of the overall domain structure of BATs and the amino acid tandem repeats responsible for specification of the target nucleotide sequence 23 . d , Map depicting global distribution of endofungal Burkholderia strains and their classification into four branches 16 . e , Phylogenetic tree of TALE-like proteins from eight endofungal Burkholderia species, and plant pathogenic Ralstonia solanaceraum and Xanthomonas sp (Supplementary Fig. 3). Phylogenetic analysis was performed using MEGA7 (see Methods for details). BAT sequences obtained in this study are highlighted in bold and GenBank accession numbers are given in brackets (Supplementary Table 6). The distribution of BATs across the four Burkholderia branches is indicted as follows: orange: Pacific branch; blue: Eurasian branch; green: African branch; red: Australian branch; grey: not detected. f , Deletion of BATs decreases the sporulation ability of R. microsporus after re-infection. Photographs and spore count of R. microsporus re-infected with B. rhizoxinica wild-type (B1WT) or BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , Δ bat3 ::Kan r , Δ bat2_bat3::Kan r , or Δ bat1::Apra r -Δbat2_bat3::Kan r ) after one week of co-cultivation. N = 3 biological replicates (3 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test ( ***p
    Figure Legend Snippet: Identification and functional analysis of predicted transcription-activator like effectors (TALEs) from endofungal Burkholderia species (BATs). a , Schematic illustration of the gene clusters encoding BAT1 (RBRH_01844), BAT2 (RBRH_01776), and BAT3 (RBRH_01777) indicated in red. b , Mode of action of TALEs from Xanthomonas sp. TALEs are secreted into plant cells via the T3SS, translocate to the nucleus, and induce expression of target genes 22 . c , Schematic representation of the overall domain structure of BATs and the amino acid tandem repeats responsible for specification of the target nucleotide sequence 23 . d , Map depicting global distribution of endofungal Burkholderia strains and their classification into four branches 16 . e , Phylogenetic tree of TALE-like proteins from eight endofungal Burkholderia species, and plant pathogenic Ralstonia solanaceraum and Xanthomonas sp (Supplementary Fig. 3). Phylogenetic analysis was performed using MEGA7 (see Methods for details). BAT sequences obtained in this study are highlighted in bold and GenBank accession numbers are given in brackets (Supplementary Table 6). The distribution of BATs across the four Burkholderia branches is indicted as follows: orange: Pacific branch; blue: Eurasian branch; green: African branch; red: Australian branch; grey: not detected. f , Deletion of BATs decreases the sporulation ability of R. microsporus after re-infection. Photographs and spore count of R. microsporus re-infected with B. rhizoxinica wild-type (B1WT) or BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , Δ bat3 ::Kan r , Δ bat2_bat3::Kan r , or Δ bat1::Apra r -Δbat2_bat3::Kan r ) after one week of co-cultivation. N = 3 biological replicates (3 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test ( ***p

    Techniques Used: Functional Assay, Expressing, Sequencing, Infection, Mutagenesis

    Viability test of Rhizopus microsporus re-infected with Burkholderia rhizoxinica transcription-activator like effector (BAT) mutant strains. a , Endosymbiont-free R. microsporus ATCC62417/S was co-incubated with B. rhizoxinica BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , and Δ bat3 ::Kan r ) for 72 hours. Co-cultures were stained with LIVE/DEAD BacLight fluorescent dyes inside the microfluidic device. Following fluorescence microscopy, the integrated density was calculated for both live (SYTO9) and dead bacteria (propidium iodide) using Fiji and the ratio (live/dead) was plotted for each morphotype (see Methods for details). N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test (*** p
    Figure Legend Snippet: Viability test of Rhizopus microsporus re-infected with Burkholderia rhizoxinica transcription-activator like effector (BAT) mutant strains. a , Endosymbiont-free R. microsporus ATCC62417/S was co-incubated with B. rhizoxinica BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , and Δ bat3 ::Kan r ) for 72 hours. Co-cultures were stained with LIVE/DEAD BacLight fluorescent dyes inside the microfluidic device. Following fluorescence microscopy, the integrated density was calculated for both live (SYTO9) and dead bacteria (propidium iodide) using Fiji and the ratio (live/dead) was plotted for each morphotype (see Methods for details). N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test (*** p

    Techniques Used: Infection, Mutagenesis, Incubation, Staining, Fluorescence, Microscopy

    Course of reinfection of Burkholderia rhizoxinica transcription-activator like effector (BAT) mutant strain into Rhizopus microsporus . a , Endosymbiont-free R. microsporus ATCC62417/S was co-incubated with B. rhizoxinica BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , and Δ bat3 ::Kan r ) for 48 hours. Bacterial cells were stained with SYTO9 prior to co-incubation. Following fluorescence microscopy at 485/498 nm (SYTO9), the integrated density (product of area and mean grey value) per bacterial cell number was calculated for both measurements using Fiji 36 , and then plotted as percent of the positive control ( R. microsporus ATCC62417/S co-incubated with wild-type B. rhizoxinica; % of B1WT). N = 10 biological replicates ± one SEM. b , Integrated density (in % of B1WT) was measured for each individual morphotype (1 – 4) following reinfection with BAT mutant strains. N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test (*** p
    Figure Legend Snippet: Course of reinfection of Burkholderia rhizoxinica transcription-activator like effector (BAT) mutant strain into Rhizopus microsporus . a , Endosymbiont-free R. microsporus ATCC62417/S was co-incubated with B. rhizoxinica BAT mutant strains (Δ bat1 ::Apra r , Δ bat2 ::Kan r , and Δ bat3 ::Kan r ) for 48 hours. Bacterial cells were stained with SYTO9 prior to co-incubation. Following fluorescence microscopy at 485/498 nm (SYTO9), the integrated density (product of area and mean grey value) per bacterial cell number was calculated for both measurements using Fiji 36 , and then plotted as percent of the positive control ( R. microsporus ATCC62417/S co-incubated with wild-type B. rhizoxinica; % of B1WT). N = 10 biological replicates ± one SEM. b , Integrated density (in % of B1WT) was measured for each individual morphotype (1 – 4) following reinfection with BAT mutant strains. N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test (*** p

    Techniques Used: Mutagenesis, Incubation, Staining, Fluorescence, Microscopy, Positive Control

    Phenotypic observations of Rhizopus microsporus co-cultivated with Burkholderia rhizoxinica wild-type or mutant strains in bacterial-fungal interaction (BFI) devices. a , Photograph and simplified illustration showing the microfluidic device in a glass petri dish. The BFI device is made of a patterned poly(dimethylsiloxane) layer bonded to glass-bottomed dish to form microchannels. The microchannels were filled with potato dextrose broth. Scale bar: 5 mm. A simplified two-dimensional representation of the design showing the narrow entry points into the microchannels which limits the number of hyphae that can enter the device 24 . b , Illustration showing the workflow for observation of BFIs. An agar plug containing endosymbiont-free R. microsporus is placed in direct contact with the microchannels. After two days of incubation, hyphae are growing inside the microchannels and SYTO9-stained bacterial strains are introduced into the microchannels via the ‘bacteria inlet’. Fungal reinfection is monitored over 48 hours and microscopic images are taken every 24 hours. c , Microscopic images of R. microsporus co-cultivated with wild-type B. rhizoxinica (stained with SYTO9) depicting four morphologically distinct types of hyphae (1: vegetative side hyphae; 2: vegetative main hyphae; 3: vegetative empty side hyphae; 4: abortive sporangiophores). Arrowheads indicate the presence of septa. Images were taken 48 hours post infection (hpi). Scale bar: 10 μm. Microscopic images of R. microsporus co-cultivated with B. rhizoxinica mutant strains are shown in Supplementary Fig. 5. d , The fungal mycelium area (in percent) of each morphotype was measured over a 48 hour time period of co-incubation in BFI devices. At time point 0, bacterial cells (B1WT: wild-type B. rhizoxinica; Δbat1: Δbat1::Apra r , Δ bat2: Δ bat2 ::Kan r , Δ bat3: Δ bat3 ::Kan r , Δ sctC: Δ sctC ::Kan r , Δ sctT: Δ sctT ::Kan r , and Δ rhiG: Δ rhiG ::Kan r ) were stained with SYTO9, added to the inlet, and co-incubated with endosymbiont-free R. microsporus . Images were taken at the time of infection (0 hpi), as well as 24 and 48 hpi. N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test ( ***p
    Figure Legend Snippet: Phenotypic observations of Rhizopus microsporus co-cultivated with Burkholderia rhizoxinica wild-type or mutant strains in bacterial-fungal interaction (BFI) devices. a , Photograph and simplified illustration showing the microfluidic device in a glass petri dish. The BFI device is made of a patterned poly(dimethylsiloxane) layer bonded to glass-bottomed dish to form microchannels. The microchannels were filled with potato dextrose broth. Scale bar: 5 mm. A simplified two-dimensional representation of the design showing the narrow entry points into the microchannels which limits the number of hyphae that can enter the device 24 . b , Illustration showing the workflow for observation of BFIs. An agar plug containing endosymbiont-free R. microsporus is placed in direct contact with the microchannels. After two days of incubation, hyphae are growing inside the microchannels and SYTO9-stained bacterial strains are introduced into the microchannels via the ‘bacteria inlet’. Fungal reinfection is monitored over 48 hours and microscopic images are taken every 24 hours. c , Microscopic images of R. microsporus co-cultivated with wild-type B. rhizoxinica (stained with SYTO9) depicting four morphologically distinct types of hyphae (1: vegetative side hyphae; 2: vegetative main hyphae; 3: vegetative empty side hyphae; 4: abortive sporangiophores). Arrowheads indicate the presence of septa. Images were taken 48 hours post infection (hpi). Scale bar: 10 μm. Microscopic images of R. microsporus co-cultivated with B. rhizoxinica mutant strains are shown in Supplementary Fig. 5. d , The fungal mycelium area (in percent) of each morphotype was measured over a 48 hour time period of co-incubation in BFI devices. At time point 0, bacterial cells (B1WT: wild-type B. rhizoxinica; Δbat1: Δbat1::Apra r , Δ bat2: Δ bat2 ::Kan r , Δ bat3: Δ bat3 ::Kan r , Δ sctC: Δ sctC ::Kan r , Δ sctT: Δ sctT ::Kan r , and Δ rhiG: Δ rhiG ::Kan r ) were stained with SYTO9, added to the inlet, and co-incubated with endosymbiont-free R. microsporus . Images were taken at the time of infection (0 hpi), as well as 24 and 48 hpi. N = 3 biological replicates (16 technical replicates) ± one SEM. One-way ANOVA with Tukey’s multiple comparison test ( ***p

    Techniques Used: Mutagenesis, Incubation, Staining, Infection

    33) Product Images from "Filter paper-based spin column method for cost-efficient DNA or RNA purification"

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203011

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Figure Legend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Techniques Used: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    34) Product Images from "Adaptable and Efficient Genome Editing by sgRNA-Cas9 Protein Co-injection into Drosophila"

    Article Title: Adaptable and Efficient Genome Editing by sgRNA-Cas9 Protein Co-injection into Drosophila

    Journal: bioRxiv

    doi: 10.1101/2020.05.07.080762

    Workflow for two-step genome editing. ( 1a ) Target sites flanking the area to be edited are identified (red, blue, geen, purple) using online tools searching for optimal targets and with minimal off-target cleavage. ( 1b ) Sequences from the selected target sites are transcribed in vitro to generate sgRNAs. ( 1c ) Cas9 protein is incubated with sgRNAs before injection into embryos. ( 1d ) Active sgRNAs that cleave embryo DNA are identified by T7 endonuclease I reactions. ( 1e ) One of the active sgRNAs is chosen for genome editing in Step 2. ( 2a ) Homology arms flanking the region of interest are cloned into the pBS- GMR-eya (shRNA) donor plasmid. In this example, the CRISPR target site (red triangle) is 5’ to the bases to be edited (black bar). ( 2b ) Embryos are injected with the repair template plasmid and RNPs composed of sgRNA and Cas9 protein. ( 2c ) Adult flies that develop from injected embryos are crossed back to the parental line. G1 progeny are screened for the DsRed marker. Positive G1 animals may have small eyes due to eya (shRNA) but these are not selected (green circle). Only positive G1 animals with normal eyes are selected (red circle). ( 2d ) These are crossed to make purebred lines and molecularly analyzed to determine if they contain the desired editing events. ( 2e ) PiggyBac transposase is expressed in the germline, either by a single cross to a transgenic line, or in this example, by embryo injection of a plasmid expressing the transposase. ( 2f ) Since the DsRed marker is dominant, adult flies developing from injected embryos that do not have red fluorescent eyes are then crossed and analyzed with molecular tests to determine whether they have precisely excised the marker gene. Only the intended genomic edit remains.
    Figure Legend Snippet: Workflow for two-step genome editing. ( 1a ) Target sites flanking the area to be edited are identified (red, blue, geen, purple) using online tools searching for optimal targets and with minimal off-target cleavage. ( 1b ) Sequences from the selected target sites are transcribed in vitro to generate sgRNAs. ( 1c ) Cas9 protein is incubated with sgRNAs before injection into embryos. ( 1d ) Active sgRNAs that cleave embryo DNA are identified by T7 endonuclease I reactions. ( 1e ) One of the active sgRNAs is chosen for genome editing in Step 2. ( 2a ) Homology arms flanking the region of interest are cloned into the pBS- GMR-eya (shRNA) donor plasmid. In this example, the CRISPR target site (red triangle) is 5’ to the bases to be edited (black bar). ( 2b ) Embryos are injected with the repair template plasmid and RNPs composed of sgRNA and Cas9 protein. ( 2c ) Adult flies that develop from injected embryos are crossed back to the parental line. G1 progeny are screened for the DsRed marker. Positive G1 animals may have small eyes due to eya (shRNA) but these are not selected (green circle). Only positive G1 animals with normal eyes are selected (red circle). ( 2d ) These are crossed to make purebred lines and molecularly analyzed to determine if they contain the desired editing events. ( 2e ) PiggyBac transposase is expressed in the germline, either by a single cross to a transgenic line, or in this example, by embryo injection of a plasmid expressing the transposase. ( 2f ) Since the DsRed marker is dominant, adult flies developing from injected embryos that do not have red fluorescent eyes are then crossed and analyzed with molecular tests to determine whether they have precisely excised the marker gene. Only the intended genomic edit remains.

    Techniques Used: In Vitro, Incubation, Injection, Clone Assay, shRNA, Plasmid Preparation, CRISPR, Marker, Transgenic Assay, Expressing

    The modified plasmid backbone for HDR editing. Shown is the transgenic marker for counterselection of imprecise HDR events. The GMR element contains 5 tandem binding sites for the transcription factor Glass fused to the Hsp70 minimal promoter. The transcript contains a shRNA stem-loop followed by an intron from the ftz gene to facilitate transcript stability. After the shRNA is processed by Drosha and Dicer, the guide RNA strand is loaded into RISC. The guide RNA is perfectly complementary to all mRNA isoforms of eya . Shown only is isoform C, and the location of the RNAi target is indicated.
    Figure Legend Snippet: The modified plasmid backbone for HDR editing. Shown is the transgenic marker for counterselection of imprecise HDR events. The GMR element contains 5 tandem binding sites for the transcription factor Glass fused to the Hsp70 minimal promoter. The transcript contains a shRNA stem-loop followed by an intron from the ftz gene to facilitate transcript stability. After the shRNA is processed by Drosha and Dicer, the guide RNA strand is loaded into RISC. The guide RNA is perfectly complementary to all mRNA isoforms of eya . Shown only is isoform C, and the location of the RNAi target is indicated.

    Techniques Used: Modification, Plasmid Preparation, Transgenic Assay, Marker, Binding Assay, shRNA

    35) Product Images from "An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization"

    Article Title: An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization

    Journal: bioRxiv

    doi: 10.1101/799833

    RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. (A) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. (B) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In (A), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p
    Figure Legend Snippet: RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. (A) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. (B) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In (A), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p

    Techniques Used: Activity Assay, Injection, Functional Assay, One-tailed Test

    36) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.
    Figure Legend Snippet: EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Techniques Used: Methylation, CpG Methylation Assay

    37) Product Images from "Selection of an Efficient AAV Vector for Robust CNS Transgene Expression"

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.007

    iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.
    Figure Legend Snippet: iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.

    Techniques Used: Selection, Expressing, Construct, Crocin Bleaching Assay, Clone Assay, Injection, Transgenic Assay, Functional Assay, Transduction, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, DNA Sequencing

    38) Product Images from "Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens"

    Article Title: Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0235222

    Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.
    Figure Legend Snippet: Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.

    Techniques Used: Polymerase Chain Reaction

    39) Product Images from "Variability within rare cell states enables multiple paths towards drug resistance"

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.996660

    Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).
    Figure Legend Snippet: Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).

    Techniques Used: Inhibition, Sequencing, Fluorescence In Situ Hybridization, Labeling, RNA Sequencing Assay, Expressing, Isolation, In Situ

    Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).
    Figure Legend Snippet: Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization, RNA Sequencing Assay, Generated, Expressing, Migration, Marker, Staining

    40) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: bioRxiv

    doi: 10.1101/815092

    High-temperature in vitro transcription does not affect antisense dsRNA by-product formation. A) Native gel electrophoresis analyses of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. B) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. C) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C vs . 50°C.
    Figure Legend Snippet: High-temperature in vitro transcription does not affect antisense dsRNA by-product formation. A) Native gel electrophoresis analyses of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. B) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. C) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C vs . 50°C.

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, Purification

    Antisense dsRNA by-product formation is template 3’ sequence dependent. A) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3’-terminal 25 bp sequence was moved to various positions within 512B template (512B-A to 512B-D). B) dsRNA immunoblot and native gel analysis of modified 512B templates (512B-A to 512B-D) using TsT7-1 at 37°C vs. 50°C. C) A chimeric template was generated in which 26 bp of the CLuc 3’-end sequence was added to the 3’-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for one hour.
    Figure Legend Snippet: Antisense dsRNA by-product formation is template 3’ sequence dependent. A) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3’-terminal 25 bp sequence was moved to various positions within 512B template (512B-A to 512B-D). B) dsRNA immunoblot and native gel analysis of modified 512B templates (512B-A to 512B-D) using TsT7-1 at 37°C vs. 50°C. C) A chimeric template was generated in which 26 bp of the CLuc 3’-end sequence was added to the 3’-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for one hour.

    Techniques Used: Sequencing, Modification, Generated, Nucleic Acid Electrophoresis

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.
    Figure Legend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, In Vitro

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    Article Snippet: .. For all DNA preparations, either the Monarch® Plasmid Miniprep Kit (New England Biolabs, Ipswich, MA, USA) or the QIAprep® Spin Miniprep kit was used (Qiagen, Venlo, Netherlands). .. DNA cleaning and/or concentrating was done using the DNA Clean and Concentrator® -5 kit (Zymo Research, Tustin, CA, USA) as needed between PCR/amplification steps, unless otherwise specified.

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: .. After confirmation of the inserts´ identities and correct orientation by sequencing (Seqlab, Göttingen, Germany), plasmid DNA isolated from E . coli DH5α (Zippy Plasmid Miniprep kit, Epigenetics) was electroporated into E . coli BL21(DE3) expression host (New England Biolabs) as above. .. The cdt V-B deletion mutant (cdt V-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( ) and plasmid DNA from strain BL21(DE3)/pET23b(+)cdt V-ABC ( ) as a template.

    Article Title: Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors
    Article Snippet: .. Cosmid DNA was obtained from each bioactive clone using a Monarch Plasmid Miniprep kit (NEB, Ipswich, MA) and sequenced by Sanger sequencing using primers targeting the flanking regions of the ScaI site on pJWC1 vector ( ). ..

    Article Title: TET enzymes control antibody production and shape the mutational landscape in germinal centre B cells
    Article Snippet: .. Plasmid DNA was extracted using the Monarch Plasmid Miniprep Kit (New England BioLabs, T1010L) as per manufacturer's instructions. .. Subsequently, Sanger sequencing was performed using the pJet1.2 F primer detailed above.

    Article Title: High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing
    Article Snippet: .. For transformation, electro-competent cells of the CF isolate were mixed with 5 μl of the purified pCM28nuc plasmid (NEB Monarch Plasmid Miniprep Kit). .. Electroporation was executed by the Ec2 program of the BIORad MicroPulser Electroporator (Pulse 2.5 kV, number of impulse 1) in a 0.2 cm electroporation cuvette.

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  • 99
    New England Biolabs monarch plasmid miniprep kit
    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin <t>miniprep</t> kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Monarch Plasmid Miniprep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch plasmid miniprep kit/product/New England Biolabs
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    monarch plasmid miniprep kit - by Bioz Stars, 2020-08
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    91
    New England Biolabs monarch pcr dna
    Serum stability profiling: (A) <t>PCR</t> amplified <t>DNA</t> and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Monarch Pcr Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch pcr dna/product/New England Biolabs
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    monarch pcr dna - by Bioz Stars, 2020-08
    91/100 stars
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    99
    New England Biolabs monarch dna gel extraction kit
    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic <t>DNA.</t> Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) <t>PCR</t> evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Monarch Dna Gel Extraction Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch dna gel extraction kit/product/New England Biolabs
    Average 99 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    monarch dna gel extraction kit - by Bioz Stars, 2020-08
    99/100 stars
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    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Journal: PLoS ONE

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    doi: 10.1371/journal.pone.0203011

    Figure Lengend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Article Snippet: Alternatively, spin columns with a conical (V-shape) bottom and a drip opening, such as a miniprep column from Qiagen , and a recent version adopted in NEB Monarch plasmid miniprep kit, can be recharged by reloading filter paper discs with a diameter of 5/16 inch (~8 mm) ( ).

    Techniques: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Journal: Frontiers in Chemistry

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    doi: 10.3389/fchem.2020.00400

    Figure Lengend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Article Snippet: Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India.

    Techniques: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Journal: Frontiers in Microbiology

    Article Title: Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior

    doi: 10.3389/fmicb.2018.01569

    Figure Lengend Snippet: CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Article Snippet: All PCR products and plasmids were purified using Monarch DNA Gel Extraction Kit (NEB, United States) and MiniBEST Plasmid Purification Kit (Takara, Japan), respectively.

    Techniques: CRISPR, Introduce, Sequencing, Polymerase Chain Reaction, Amplification