hiscribe  (New England Biolabs)


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    New England Biolabs hiscribe
    Hiscribe, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiscribe/product/New England Biolabs
    Average 97 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    hiscribe - by Bioz Stars, 2022-09
    97/100 stars

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    New England Biolabs hiscribe t7 quick high yield rna synthesis kit
    The detection limit of RRCd for SARS-CoV-2 VOCs. The detection limit of the targeted regions in VOCs were determined by using 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 and 5 copies of pseudovirus <t>RNA</t> per reaction, respectively. The dsDNA derived from the pseudovirus RNA was incubated with Cas12a, ssDNA probe, and crRNA at 37 °C for 60 min (fluorescence-based readouts) or 30 min (lateral-flow readouts), respectively. The relative fluorescence intensity (F/F 0 ) was calculated as the fluorescence signal versus the starting signal (left panel). Correspondingly, the fluorescence signal at the 30 min-reaction was quantified (middle panel). The positive samples of lateral-flow readouts (right panel) are indicated as blue by the T-line quantification as described. Data are presented as mean ± S.D. from at least three independent experiments. For the dilutions with 10 and 5 copies per reaction, at least five independent experiments were conducted. A . LoD determination of RRCd for E gene. B-E . LoD determinations of RRCd for N501 (B), Y501 (C), T478 (D) and K478 (E) in S gene, respectively. NC, negative control using the RNase-free water.
    Hiscribe T7 Quick High Yield Rna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiscribe t7 quick high yield rna synthesis kit/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiscribe t7 quick high yield rna synthesis kit - by Bioz Stars, 2022-09
    97/100 stars
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    94
    New England Biolabs hiscribetm t7 arca mrna kit
    <t>B.1.351-LNP-mRNA</t> and <t>B.1.617-LNP-mRNA</t> shown in vivo to protect efficacy against the challenge of replication competent authentic SARS-CoV-2 and variant viruses (A) Schematic of authentic virus challenge experiments on mRNA-LNP-vaccinated mice. hACE2-K18 mice were separated randomly and received 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). One week after boost (day 28), the mRNA-LNP-vaccinated, and control mice were distributed into three groups and challenged with WA-1, Beta, and Delta authentic live virus. Survival, body conditions, and weights of mice were monitored daily for 10 consecutive days. (B) A numeric summary of the number of hACE2-K18 mice vaccinated with WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA and challenged with three different authentic virus WA01, Beta (B.1.351), and Delta (B.1.617.2). (C) Body weight curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). (D) Survival curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, or B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p
    Hiscribetm T7 Arca Mrna Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiscribetm t7 arca mrna kit/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiscribetm t7 arca mrna kit - by Bioz Stars, 2022-09
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    99
    New England Biolabs hiscribe t7 high yield rna synthesis kit
    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) on a SARS-CoV-2 synthetic calibration standard. ( A ) A synthetic calibration standard for SARS-CoV-2 N2 <t>RNA</t> was synthesized including a 7 nt divergent sequence (in red), maintaining all other LAMP primer binding sites and identical GC content. ( B ) RT-LAMP using COV-ID N2 primers was carried out on indicated amounts of synthetic calibration standard (SCS) RNA, showing rapid amplification down to picogram quantities of added template. ( C ) Total number of reads per barcode in COV-ID pool obtained by including (+) or omitting (−) the N2 synthetic calibration standard. ( D ) Spurious COV-ID signal for the N2 amplicon in negative control samples after normalization either to the STATH control (in absence of the synthetic calibration standard) or to the SCS. Bars indicate the mean + standard deviation.
    Hiscribe T7 High Yield Rna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiscribe t7 high yield rna synthesis kit/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    hiscribe t7 high yield rna synthesis kit - by Bioz Stars, 2022-09
    99/100 stars
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    Image Search Results


    The detection limit of RRCd for SARS-CoV-2 VOCs. The detection limit of the targeted regions in VOCs were determined by using 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 and 5 copies of pseudovirus RNA per reaction, respectively. The dsDNA derived from the pseudovirus RNA was incubated with Cas12a, ssDNA probe, and crRNA at 37 °C for 60 min (fluorescence-based readouts) or 30 min (lateral-flow readouts), respectively. The relative fluorescence intensity (F/F 0 ) was calculated as the fluorescence signal versus the starting signal (left panel). Correspondingly, the fluorescence signal at the 30 min-reaction was quantified (middle panel). The positive samples of lateral-flow readouts (right panel) are indicated as blue by the T-line quantification as described. Data are presented as mean ± S.D. from at least three independent experiments. For the dilutions with 10 and 5 copies per reaction, at least five independent experiments were conducted. A . LoD determination of RRCd for E gene. B-E . LoD determinations of RRCd for N501 (B), Y501 (C), T478 (D) and K478 (E) in S gene, respectively. NC, negative control using the RNase-free water.

    Journal: medRxiv

    Article Title: RT-RPA-Cas12a-based discrimination of SARS-CoV-2 variants of concern

    doi: 10.1101/2022.05.11.22274884

    Figure Lengend Snippet: The detection limit of RRCd for SARS-CoV-2 VOCs. The detection limit of the targeted regions in VOCs were determined by using 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 and 5 copies of pseudovirus RNA per reaction, respectively. The dsDNA derived from the pseudovirus RNA was incubated with Cas12a, ssDNA probe, and crRNA at 37 °C for 60 min (fluorescence-based readouts) or 30 min (lateral-flow readouts), respectively. The relative fluorescence intensity (F/F 0 ) was calculated as the fluorescence signal versus the starting signal (left panel). Correspondingly, the fluorescence signal at the 30 min-reaction was quantified (middle panel). The positive samples of lateral-flow readouts (right panel) are indicated as blue by the T-line quantification as described. Data are presented as mean ± S.D. from at least three independent experiments. For the dilutions with 10 and 5 copies per reaction, at least five independent experiments were conducted. A . LoD determination of RRCd for E gene. B-E . LoD determinations of RRCd for N501 (B), Y501 (C), T478 (D) and K478 (E) in S gene, respectively. NC, negative control using the RNase-free water.

    Article Snippet: The crRNAs were transcribed using HiScribe T7 High Yield RNA Synthesis kit (Cat# E2050S, New England Biolabs) at 37 °C for 16 h. Then, the crRNAs were purified using RNA Clean and Concentrator kit (Cat# R1017, Zymo Research).

    Techniques: Derivative Assay, Incubation, Fluorescence, Negative Control

    Detection and discrimination of SARS-CoV-2 VOCs by RRCd. Schematic overview of RRCd workflow. (I) SARS-CoV-2 samples were released by automatic extraction (∼10 min) or using RNA release chemical agents without extraction (∼5 min). (II) The viral RNA was reverse transcribed into the dsDNA and amplified by RPA. (III) Cas12a was guided to target the dsDNA by the specific crRNA, thereby activated its trans -cleavage activity to the ssDNA probes in the system. The results could be quantified by fluorescence detection of the cleaved FQ-ssDNA (III a), or visualized by lateral-flow strip using the FB-ssDNA probe (III b). RRCd in discrimination of SARS-CoV-2 VOCs with lateral-flow readout is highlighted on the right panel. The target gene from SARS-CoV-2 variant was shown as the template, and it was detected by either crRNA-VOC or crRNA-WT, leading to the positive (upper right panel) or negative (lower right panel) test result, respectively. dsDNA, double-strand DNA; ssDNA, single-strand DNA; FQ, fluorophore (FAM)-quencher (BHQ1); FB, FITC-Biotin; PAM, protospacer adjacent motif; VOC, variant of concern; WT, wild type; GNP, gold nanoparticle; gt, goat.

    Journal: medRxiv

    Article Title: RT-RPA-Cas12a-based discrimination of SARS-CoV-2 variants of concern

    doi: 10.1101/2022.05.11.22274884

    Figure Lengend Snippet: Detection and discrimination of SARS-CoV-2 VOCs by RRCd. Schematic overview of RRCd workflow. (I) SARS-CoV-2 samples were released by automatic extraction (∼10 min) or using RNA release chemical agents without extraction (∼5 min). (II) The viral RNA was reverse transcribed into the dsDNA and amplified by RPA. (III) Cas12a was guided to target the dsDNA by the specific crRNA, thereby activated its trans -cleavage activity to the ssDNA probes in the system. The results could be quantified by fluorescence detection of the cleaved FQ-ssDNA (III a), or visualized by lateral-flow strip using the FB-ssDNA probe (III b). RRCd in discrimination of SARS-CoV-2 VOCs with lateral-flow readout is highlighted on the right panel. The target gene from SARS-CoV-2 variant was shown as the template, and it was detected by either crRNA-VOC or crRNA-WT, leading to the positive (upper right panel) or negative (lower right panel) test result, respectively. dsDNA, double-strand DNA; ssDNA, single-strand DNA; FQ, fluorophore (FAM)-quencher (BHQ1); FB, FITC-Biotin; PAM, protospacer adjacent motif; VOC, variant of concern; WT, wild type; GNP, gold nanoparticle; gt, goat.

    Article Snippet: The crRNAs were transcribed using HiScribe T7 High Yield RNA Synthesis kit (Cat# E2050S, New England Biolabs) at 37 °C for 16 h. Then, the crRNAs were purified using RNA Clean and Concentrator kit (Cat# R1017, Zymo Research).

    Techniques: Amplification, Recombinase Polymerase Amplification, Activity Assay, Fluorescence, Stripping Membranes, Variant Assay

    B.1.351-LNP-mRNA and B.1.617-LNP-mRNA shown in vivo to protect efficacy against the challenge of replication competent authentic SARS-CoV-2 and variant viruses (A) Schematic of authentic virus challenge experiments on mRNA-LNP-vaccinated mice. hACE2-K18 mice were separated randomly and received 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). One week after boost (day 28), the mRNA-LNP-vaccinated, and control mice were distributed into three groups and challenged with WA-1, Beta, and Delta authentic live virus. Survival, body conditions, and weights of mice were monitored daily for 10 consecutive days. (B) A numeric summary of the number of hACE2-K18 mice vaccinated with WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA and challenged with three different authentic virus WA01, Beta (B.1.351), and Delta (B.1.617.2). (C) Body weight curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). (D) Survival curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, or B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: B.1.351-LNP-mRNA and B.1.617-LNP-mRNA shown in vivo to protect efficacy against the challenge of replication competent authentic SARS-CoV-2 and variant viruses (A) Schematic of authentic virus challenge experiments on mRNA-LNP-vaccinated mice. hACE2-K18 mice were separated randomly and received 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). One week after boost (day 28), the mRNA-LNP-vaccinated, and control mice were distributed into three groups and challenged with WA-1, Beta, and Delta authentic live virus. Survival, body conditions, and weights of mice were monitored daily for 10 consecutive days. (B) A numeric summary of the number of hACE2-K18 mice vaccinated with WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA and challenged with three different authentic virus WA01, Beta (B.1.351), and Delta (B.1.617.2). (C) Body weight curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). (D) Survival curves of WT-LNP mRNA-, B.1.351-LNP-mRNA-, or B.1.617-LNP-mRNA-vaccinated, and control hACE2 transgenic mice under lethal challenges with different authentic virus WA-01 (left), Beta (middle), and Delta (right). Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: In Vivo, Variant Assay, Mouse Assay, Transgenic Assay

    Single-cell transcriptomics of variant-specific LNP-mRNA-vaccinated animals (A) UMAP visualizations of all 141,729 cells pooled across samples and conditions. Cells are color labeled by vaccine, concentration, and unsupervised clustering in each panel, top to bottom. Clusters are labeled by cell types that were assigned based on the expression of cell type-specific markers. (B) UMAP heatmaps of the expression of major cell type-specific markers across all cells. (C) Heatmap of differentially expressed genes (DEGs) across indicated cell types. Differential expression analyses were performed using Wilcoxon rank-sum test for each cell type versus all other cells, and the heatmap includes the 10 DEGs from each analysis (absolute log 2 -FC > 4, q

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: Single-cell transcriptomics of variant-specific LNP-mRNA-vaccinated animals (A) UMAP visualizations of all 141,729 cells pooled across samples and conditions. Cells are color labeled by vaccine, concentration, and unsupervised clustering in each panel, top to bottom. Clusters are labeled by cell types that were assigned based on the expression of cell type-specific markers. (B) UMAP heatmaps of the expression of major cell type-specific markers across all cells. (C) Heatmap of differentially expressed genes (DEGs) across indicated cell types. Differential expression analyses were performed using Wilcoxon rank-sum test for each cell type versus all other cells, and the heatmap includes the 10 DEGs from each analysis (absolute log 2 -FC > 4, q

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Single-cell Transcriptomics, Variant Assay, Labeling, Concentration Assay, Expressing

    B.1.351-LNP-mRNA and B.1.617-LNP-mRNA elicit robust binding and pseudovirus-neutralizing antibody response against all three variants in mice (A) Serum ELISA titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against RBD from three different spikes (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (B) Serum ELISA titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against ECD from three different spikes (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (C) Serum neutralization titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against three pseudoviruses (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (D and E) Direct comparison of serum ELISA (D) and neutralization (E) titers of animals boosted by WT, B.1.351-LNP-mRNA, and B.1.617-LNP-mRNA against WT, B.1.351, and B.1.617 spikes or pseudoviruses of SARS-CoV-2.F.Heatmap of neutralization titers of animals vaccinated with all three LNP-mRNAs, against three pseudoviruses (WT, B.1.351, and B.1.617) of SARS-CoV-2. G, correlation X-Y scatterplots of ELISA and neutralization titers between ELISA ECD log 10 AUC versus neutralization log 10 IC 50 for all vaccine groups. Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: B.1.351-LNP-mRNA and B.1.617-LNP-mRNA elicit robust binding and pseudovirus-neutralizing antibody response against all three variants in mice (A) Serum ELISA titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against RBD from three different spikes (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (B) Serum ELISA titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against ECD from three different spikes (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (C) Serum neutralization titers of animals vaccinated with B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA (bottom) against three pseudoviruses (WT, B.1.351, and B.1.617) of SARS-CoV-2 (n = 6). (D and E) Direct comparison of serum ELISA (D) and neutralization (E) titers of animals boosted by WT, B.1.351-LNP-mRNA, and B.1.617-LNP-mRNA against WT, B.1.351, and B.1.617 spikes or pseudoviruses of SARS-CoV-2.F.Heatmap of neutralization titers of animals vaccinated with all three LNP-mRNAs, against three pseudoviruses (WT, B.1.351, and B.1.617) of SARS-CoV-2. G, correlation X-Y scatterplots of ELISA and neutralization titers between ELISA ECD log 10 AUC versus neutralization log 10 IC 50 for all vaccine groups. Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    VDJ repertoire and clonal analyses of B cell and T cell populations from variant-specific LNP-mRNA-vaccinated animals (A) Clonal composition bar plot depicting proportion of the BCR repertoire occupied by the clones of a given size for all samples in the single-cell BCR-seq dataset. (B) Bar plot of Chao1 indices for each condition for repertoires in the single cell BCR-seq dataset (n = 6 for each group). (C) Clonal composition bar plot depicting proportion of the TCR repertoire occupied by the clones of a given size for all samples in the single-cell TCR-seq dataset. (D) Bar plot of unique clonotypes for each for repertoires in the single-cell TCR-seq. (E) Circos plots of V-J clonotype distribution for single-cell BCR-seq dataset (left) and single cell TCR-seq dataset (right). The 20 most abundant V-J combinations are shown for pooled vaccination group. (F) Clonal composition bar plot depicting proportion of the BCR repertoire occupied by the clones of a given size for all samples in the bulk BCR-seq dataset (left) and bulk TCR-seq dataset (right). (G) Bar plots depicting relative abundances of IGH, IGK, IGL, TRA, TRB, and TRD clonotypes within specific frequency ranges in the bulk BCR/TCR-seq data from different tissues of different vaccination groups. Relative abundances are presented for individual and grouped samples in (E) and (F), respectively. (H) Bar plots of the effective clone numbers (true-diversity estimates) for selected BCR and TCR chain repertoires in the bulk TCR-seq dataset across vaccination and tissue groups. Note that for the single-cell BCR/TCR-seq datasets, n = 6 samples for the PBS and n = 3 for WA-1 1 μg, WA-1 10 μg, B.1.351 1 μg, B.1.351, B.1.617 1 μg, and B.1.617 10 μg groups. For the bulk BCR/TCR-seq datasets, n = 4 PBS samples, and n = 3 for B.1.351 1 μg, B.1.351, B.1.617 1 μg, and B.1.617 10 μg groups. Statistics for (F) and (G) were performed using two-way ANOVA with Dunnet’s multiple comparison test. Statistical significance labels: n.s., not significant; ∗p

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: VDJ repertoire and clonal analyses of B cell and T cell populations from variant-specific LNP-mRNA-vaccinated animals (A) Clonal composition bar plot depicting proportion of the BCR repertoire occupied by the clones of a given size for all samples in the single-cell BCR-seq dataset. (B) Bar plot of Chao1 indices for each condition for repertoires in the single cell BCR-seq dataset (n = 6 for each group). (C) Clonal composition bar plot depicting proportion of the TCR repertoire occupied by the clones of a given size for all samples in the single-cell TCR-seq dataset. (D) Bar plot of unique clonotypes for each for repertoires in the single-cell TCR-seq. (E) Circos plots of V-J clonotype distribution for single-cell BCR-seq dataset (left) and single cell TCR-seq dataset (right). The 20 most abundant V-J combinations are shown for pooled vaccination group. (F) Clonal composition bar plot depicting proportion of the BCR repertoire occupied by the clones of a given size for all samples in the bulk BCR-seq dataset (left) and bulk TCR-seq dataset (right). (G) Bar plots depicting relative abundances of IGH, IGK, IGL, TRA, TRB, and TRD clonotypes within specific frequency ranges in the bulk BCR/TCR-seq data from different tissues of different vaccination groups. Relative abundances are presented for individual and grouped samples in (E) and (F), respectively. (H) Bar plots of the effective clone numbers (true-diversity estimates) for selected BCR and TCR chain repertoires in the bulk TCR-seq dataset across vaccination and tissue groups. Note that for the single-cell BCR/TCR-seq datasets, n = 6 samples for the PBS and n = 3 for WA-1 1 μg, WA-1 10 μg, B.1.351 1 μg, B.1.351, B.1.617 1 μg, and B.1.617 10 μg groups. For the bulk BCR/TCR-seq datasets, n = 4 PBS samples, and n = 3 for B.1.351 1 μg, B.1.351, B.1.617 1 μg, and B.1.617 10 μg groups. Statistics for (F) and (G) were performed using two-way ANOVA with Dunnet’s multiple comparison test. Statistical significance labels: n.s., not significant; ∗p

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Variant Assay, Clone Assay

    B.1.351-LNP-mRNA and B.1.617-LNP-mRNA induced S protein-specific T cell response (A–C) Percentage of CD8 + T cells expressing IFN-γ (A), TNF-α (B), and IL-2 (C) in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. (D) Percentage of CD4 + T cells expressing IFN-γ in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. B.1.351-LNP-mRNA and B.1.617-LNP-mRNA induced S protein-specific polyfunctional CD8 and CD4 T cells. (E-H) Percentage of CD8+ T cells expressing both IFN-γ and TNFα (E), both IFN-γ and IL-2 (F), TNFα and IL-2 (G), in response to stimulation of S peptide pools (n = 3). Percentage of CD4+ T cells expressing both IFN-γ and TNFα in response to stimulation of S peptide pools (H). Left panels, representative flow plots; right panels, dot-bar plots for statistics of the left panels. (H) Percentage of CD4 + T cells expressing both IFN-γ and TNF-α in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: B.1.351-LNP-mRNA and B.1.617-LNP-mRNA induced S protein-specific T cell response (A–C) Percentage of CD8 + T cells expressing IFN-γ (A), TNF-α (B), and IL-2 (C) in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. (D) Percentage of CD4 + T cells expressing IFN-γ in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. B.1.351-LNP-mRNA and B.1.617-LNP-mRNA induced S protein-specific polyfunctional CD8 and CD4 T cells. (E-H) Percentage of CD8+ T cells expressing both IFN-γ and TNFα (E), both IFN-γ and IL-2 (F), TNFα and IL-2 (G), in response to stimulation of S peptide pools (n = 3). Percentage of CD4+ T cells expressing both IFN-γ and TNFα in response to stimulation of S peptide pools (H). Left panels, representative flow plots; right panels, dot-bar plots for statistics of the left panels. (H) Percentage of CD4 + T cells expressing both IFN-γ and TNF-α in response to stimulation of S -pep tide pools (n = 3). Left: representative flow plots; right: dot-bar plots for statistics on the left. Note that in this figure, each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Expressing

    Overview of the primary experimental design and the B and T cell responses induced by WT-LNP-mRNA vaccination against SARS-CoV-2 WT, B.1.351, and B.1.617 spikes in mice (A) Schematic of the designs of three variant-specific LNP-mRNA vaccine candidates. Functional elements are shown in the spike mRNA and translated protein of SARS-CoV-2 WT, B.1.351, and B.1.617 spikes, including protein domains, HexaPro, and variant-specific mutations. (B) 3D structure highlighting certain variant-specific mutations in B.1.351 and B.1.617 spikes. Distribution of mutations of B.1.351 and B.1.617 are shown in the structure of SARS-CoV-2 (PDB: 6VSB ). Mutations of B.1.351 and B.1.617 are shown as spheres, except for those in the unstructured loop regions. Certain mutations are not visible in the structure, as they fall into floppy regions of spike. (C) Graphical representation of B.1.351-LNP-mRNA complex and B.1.617-LNP-mRNA complex formation. The spike mRNAs of B.1.351 and B.1.617 are encapsulated by LNP via NanoAssemblr Ignite. The size and encapsulation rate of the mRNA-LNP complex were measured by dynamic light scatter (DLS) and Ribogreen assay, respectively. (D) After electroporated into 293FT cells, in vitro expression of B.1.351-spike or B.1.617-spike mRNA were detected by flow cytometry using the human ACE2-Fc fusion protein and PE- anti -Fc antibody. (E and F) DLS (E) and TEM (F) of size and monodispersity characterization of LNP-mRNAs. (G) Schematic of overall design of primary experiments. Six- to 8-week-old C57BL/6Ncr mice (B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA, n = 6 mice per group; WT-LNP-mRNA, n = 4 mice; PBS, n = 9) received 1 or 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). Blood was collected twice, 2 weeks post-prime and -boost. The binding and pseudovirus-neutralizing antibody responses induced by LNP-mRNA were evaluated by ELISA and neutralization assay. Mice were euthanized at day 40. The spleen, lymph node, and blood samples were collected to analyze immune responses by flow cytometry, bulk BCR, and TCR profiling and single-cell profiling. (H and I) Serum ELISA titers of WT-LNP mRNA-vaccinated animals (n = 4). Serum antibody titer as area under curve (AUC) of log 10 -transformed curve (1og 10 AUC) to spike RBDs (H) and ECDs (I) of SARS-CoV-2 WT, B.1.351, and B.1.617. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. (J) Serum neutralization titers of WT-LNP mRNA-vaccinated animals (n = 4). Cross neutralization of SARS-CoV-2 WT, B.1.351, or B.1.617 pseudovirus infection of ACE2-overexpressed 293T cells. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. (K and L) T cell response of WT-LNP mRNA-vaccinated animals (n = 4). CD8 + (K) and CD4 + (L) T cell responses were measured by intracellular cytokine staining 6 h after addition of BFA. The unpaired parametric t test was used to evaluate the statistical significance. Note that in this figure each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: Overview of the primary experimental design and the B and T cell responses induced by WT-LNP-mRNA vaccination against SARS-CoV-2 WT, B.1.351, and B.1.617 spikes in mice (A) Schematic of the designs of three variant-specific LNP-mRNA vaccine candidates. Functional elements are shown in the spike mRNA and translated protein of SARS-CoV-2 WT, B.1.351, and B.1.617 spikes, including protein domains, HexaPro, and variant-specific mutations. (B) 3D structure highlighting certain variant-specific mutations in B.1.351 and B.1.617 spikes. Distribution of mutations of B.1.351 and B.1.617 are shown in the structure of SARS-CoV-2 (PDB: 6VSB ). Mutations of B.1.351 and B.1.617 are shown as spheres, except for those in the unstructured loop regions. Certain mutations are not visible in the structure, as they fall into floppy regions of spike. (C) Graphical representation of B.1.351-LNP-mRNA complex and B.1.617-LNP-mRNA complex formation. The spike mRNAs of B.1.351 and B.1.617 are encapsulated by LNP via NanoAssemblr Ignite. The size and encapsulation rate of the mRNA-LNP complex were measured by dynamic light scatter (DLS) and Ribogreen assay, respectively. (D) After electroporated into 293FT cells, in vitro expression of B.1.351-spike or B.1.617-spike mRNA were detected by flow cytometry using the human ACE2-Fc fusion protein and PE- anti -Fc antibody. (E and F) DLS (E) and TEM (F) of size and monodispersity characterization of LNP-mRNAs. (G) Schematic of overall design of primary experiments. Six- to 8-week-old C57BL/6Ncr mice (B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA, n = 6 mice per group; WT-LNP-mRNA, n = 4 mice; PBS, n = 9) received 1 or 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). Blood was collected twice, 2 weeks post-prime and -boost. The binding and pseudovirus-neutralizing antibody responses induced by LNP-mRNA were evaluated by ELISA and neutralization assay. Mice were euthanized at day 40. The spleen, lymph node, and blood samples were collected to analyze immune responses by flow cytometry, bulk BCR, and TCR profiling and single-cell profiling. (H and I) Serum ELISA titers of WT-LNP mRNA-vaccinated animals (n = 4). Serum antibody titer as area under curve (AUC) of log 10 -transformed curve (1og 10 AUC) to spike RBDs (H) and ECDs (I) of SARS-CoV-2 WT, B.1.351, and B.1.617. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. (J) Serum neutralization titers of WT-LNP mRNA-vaccinated animals (n = 4). Cross neutralization of SARS-CoV-2 WT, B.1.351, or B.1.617 pseudovirus infection of ACE2-overexpressed 293T cells. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. (K and L) T cell response of WT-LNP mRNA-vaccinated animals (n = 4). CD8 + (K) and CD4 + (L) T cell responses were measured by intracellular cytokine staining 6 h after addition of BFA. The unpaired parametric t test was used to evaluate the statistical significance. Note that in this figure each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Mouse Assay, Variant Assay, Functional Assay, In Vitro, Expressing, Flow Cytometry, Transmission Electron Microscopy, Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Transformation Assay, Infection, Staining

    Single-cell analysis of activated B cell and CD8 T cell populations with gene expression signatures of variant-specific LNP-mRNA-vaccinated animals (A) Volcano plots of differential expression (DE) analyses for each vaccination group versus PBS in B cells. Analyses were performed using quasi-likelihood F tests of scRNA-seq data fitted with gamma-Poisson generalized linear models. (B) Network plots of clustered terms from pathway analyses of upregulated genes in the indicated B cell DE analysis. Pathway enrichment analyses were performed by gProfiler2, and significantly enriched pathways were clustered with Leiden algorithm. Pathway clusters (supra-pathways) are labeled by their most significant member term along with its enrichment q value. The top five supra-pathways are shown for each plot. (C) Expression heatmaps of DE genes from selected upregulated supra-pathways in B cell DE analysis. Single-cell expression values were scaled and then averaged across vaccination groups. (D) Volcano plots of DE analyses for each vaccination group versus PBS in CD8 T cells. Analyses were performed using quasi-likelihood F tests of scRNA-seq data fitted with gamma-Poisson generalized linear models. (E) Network plots of clustered terms from pathway analyses of upregulated genes in the indicated in CD8 T cell DE analysis. Pathway enrichment analyses were performed by gProfiler2, and significantly enriched pathways were clustered with Leiden algorithm. Pathway clusters (supra-pathways) are labeled by their most significant member term along with its enrichment q value. The top five supra-pathways are shown for each plot. (F) Expression heatmaps of DE genes from selected upregulated supra-pathways in CD8 T cell DE analysis. Single-cell expression values were scaled and then averaged across vaccination groups. See also Figures S7–S9 .

    Journal: Cell Reports Medicine

    Article Title: Variant-specific vaccination induces systems immune responses and potent in vivo protection against SARS-CoV-2

    doi: 10.1016/j.xcrm.2022.100634

    Figure Lengend Snippet: Single-cell analysis of activated B cell and CD8 T cell populations with gene expression signatures of variant-specific LNP-mRNA-vaccinated animals (A) Volcano plots of differential expression (DE) analyses for each vaccination group versus PBS in B cells. Analyses were performed using quasi-likelihood F tests of scRNA-seq data fitted with gamma-Poisson generalized linear models. (B) Network plots of clustered terms from pathway analyses of upregulated genes in the indicated B cell DE analysis. Pathway enrichment analyses were performed by gProfiler2, and significantly enriched pathways were clustered with Leiden algorithm. Pathway clusters (supra-pathways) are labeled by their most significant member term along with its enrichment q value. The top five supra-pathways are shown for each plot. (C) Expression heatmaps of DE genes from selected upregulated supra-pathways in B cell DE analysis. Single-cell expression values were scaled and then averaged across vaccination groups. (D) Volcano plots of DE analyses for each vaccination group versus PBS in CD8 T cells. Analyses were performed using quasi-likelihood F tests of scRNA-seq data fitted with gamma-Poisson generalized linear models. (E) Network plots of clustered terms from pathway analyses of upregulated genes in the indicated in CD8 T cell DE analysis. Pathway enrichment analyses were performed by gProfiler2, and significantly enriched pathways were clustered with Leiden algorithm. Pathway clusters (supra-pathways) are labeled by their most significant member term along with its enrichment q value. The top five supra-pathways are shown for each plot. (F) Expression heatmaps of DE genes from selected upregulated supra-pathways in CD8 T cell DE analysis. Single-cell expression values were scaled and then averaged across vaccination groups. See also Figures S7–S9 .

    Article Snippet: A sequence-optimized mRNA encoding B.1.351 variant (6 P) or B.1.617 variant (6P) protein was synthesized in vitro using an HiscribeTM T7 ARCA mRNA Kit (with tailing) (NEB), with 50% replacement of uridine by N1-methyl-pseudouridine.

    Techniques: Single-cell Analysis, Expressing, Variant Assay, Labeling

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) on a SARS-CoV-2 synthetic calibration standard. ( A ) A synthetic calibration standard for SARS-CoV-2 N2 RNA was synthesized including a 7 nt divergent sequence (in red), maintaining all other LAMP primer binding sites and identical GC content. ( B ) RT-LAMP using COV-ID N2 primers was carried out on indicated amounts of synthetic calibration standard (SCS) RNA, showing rapid amplification down to picogram quantities of added template. ( C ) Total number of reads per barcode in COV-ID pool obtained by including (+) or omitting (−) the N2 synthetic calibration standard. ( D ) Spurious COV-ID signal for the N2 amplicon in negative control samples after normalization either to the STATH control (in absence of the synthetic calibration standard) or to the SCS. Bars indicate the mean + standard deviation.

    Journal: eLife

    Article Title: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    doi: 10.7554/eLife.69949

    Figure Lengend Snippet: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) on a SARS-CoV-2 synthetic calibration standard. ( A ) A synthetic calibration standard for SARS-CoV-2 N2 RNA was synthesized including a 7 nt divergent sequence (in red), maintaining all other LAMP primer binding sites and identical GC content. ( B ) RT-LAMP using COV-ID N2 primers was carried out on indicated amounts of synthetic calibration standard (SCS) RNA, showing rapid amplification down to picogram quantities of added template. ( C ) Total number of reads per barcode in COV-ID pool obtained by including (+) or omitting (−) the N2 synthetic calibration standard. ( D ) Spurious COV-ID signal for the N2 amplicon in negative control samples after normalization either to the STATH control (in absence of the synthetic calibration standard) or to the SCS. Bars indicate the mean + standard deviation.

    Article Snippet: To prepare the in vitro transcription template for SARS-CoV-2 N2 synthetic calibration standard RNA, we performed RT-PCR using Power SYBR RNA-to-Ct kit (Thermo Cat. 4389986) of heat inactivated SARS-CoV-2 (BEI Resources Cat. NR-52286) using the following primers: N2-B3 and N2-spike-T7 S. PCR product was purified and used as a template for in vitro transcription using HiScribe T7 transcription kit (NEB Cat. E2040S).

    Techniques: Amplification, Synthesized, Sequencing, Binding Assay, Negative Control, Standard Deviation

    Clinical validation of COV-ID on RNA from nasopharyngeal (NP) swabs. ( A ) COV-ID on RNA from 120 patient-derived NP swabs. COV-ID for SARS-CoV-2 and ACTB was performed using 10 unique LAMP barcodes. Pools of 10 reactions were PCR amplified and sequenced to a minimum depth of 1,000 reads. Scatterplot shows SARS/(SCS +1) ratio against mean N1/N2 Ct value from RT-qPCR assays. Red circles represent samples with Ct

    Journal: eLife

    Article Title: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    doi: 10.7554/eLife.69949

    Figure Lengend Snippet: Clinical validation of COV-ID on RNA from nasopharyngeal (NP) swabs. ( A ) COV-ID on RNA from 120 patient-derived NP swabs. COV-ID for SARS-CoV-2 and ACTB was performed using 10 unique LAMP barcodes. Pools of 10 reactions were PCR amplified and sequenced to a minimum depth of 1,000 reads. Scatterplot shows SARS/(SCS +1) ratio against mean N1/N2 Ct value from RT-qPCR assays. Red circles represent samples with Ct

    Article Snippet: To prepare the in vitro transcription template for SARS-CoV-2 N2 synthetic calibration standard RNA, we performed RT-PCR using Power SYBR RNA-to-Ct kit (Thermo Cat. 4389986) of heat inactivated SARS-CoV-2 (BEI Resources Cat. NR-52286) using the following primers: N2-B3 and N2-spike-T7 S. PCR product was purified and used as a template for in vitro transcription using HiScribe T7 transcription kit (NEB Cat. E2040S).

    Techniques: Derivative Assay, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

    COV-ID multiplex detection of SARS-COV-2 and Influenza A. ( A ) TCEP/EDTA treated saliva was spiked with indicated amounts of BEI heat-inactivated SARS-CoV-2 or H1N1 influenza A RNA to the indicated concentration of virions/genomes per µL. One microliter of saliva was used for COV-ID reactions. ( B ) COV-ID was performed in two independent experiments on saliva samples from the matrix shown in ( A ) in the presence of 20 femtograms N2 synthetic calibration standard (SCS) using N2, influenza ( MacKay et al., 2020 ) and STATH COV-ID primers. N2/(SCS +1) and influenza/(STATH +1) read ratios are displayed with bars showing median ± interquartile range. Circles represent individual biological replicates. Samples were considered positive for a given sequence if the associated read ratio was greater than 2x the maximum value in the control saliva samples. ( C ) Heatmaps of SARS-CoV-2 (left) or H1N1 (right) COV-ID signal in multiplex reaction. Individual data points are from ( B ). The heatmap color represents the mean of the percentage of viral reads in each sample.

    Journal: eLife

    Article Title: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    doi: 10.7554/eLife.69949

    Figure Lengend Snippet: COV-ID multiplex detection of SARS-COV-2 and Influenza A. ( A ) TCEP/EDTA treated saliva was spiked with indicated amounts of BEI heat-inactivated SARS-CoV-2 or H1N1 influenza A RNA to the indicated concentration of virions/genomes per µL. One microliter of saliva was used for COV-ID reactions. ( B ) COV-ID was performed in two independent experiments on saliva samples from the matrix shown in ( A ) in the presence of 20 femtograms N2 synthetic calibration standard (SCS) using N2, influenza ( MacKay et al., 2020 ) and STATH COV-ID primers. N2/(SCS +1) and influenza/(STATH +1) read ratios are displayed with bars showing median ± interquartile range. Circles represent individual biological replicates. Samples were considered positive for a given sequence if the associated read ratio was greater than 2x the maximum value in the control saliva samples. ( C ) Heatmaps of SARS-CoV-2 (left) or H1N1 (right) COV-ID signal in multiplex reaction. Individual data points are from ( B ). The heatmap color represents the mean of the percentage of viral reads in each sample.

    Article Snippet: To prepare the in vitro transcription template for SARS-CoV-2 N2 synthetic calibration standard RNA, we performed RT-PCR using Power SYBR RNA-to-Ct kit (Thermo Cat. 4389986) of heat inactivated SARS-CoV-2 (BEI Resources Cat. NR-52286) using the following primers: N2-B3 and N2-spike-T7 S. PCR product was purified and used as a template for in vitro transcription using HiScribe T7 transcription kit (NEB Cat. E2040S).

    Techniques: Multiplex Assay, Concentration Assay, Sequencing

    COV-ID on saliva collected on paper. ( A ) Scheme for COV-ID on viral RNA absorbed on paper. ( B ) PCR reactions from paper samples immersed in water with indicated viral concentrations then amplified with N2 COV-ID primers. ( C ) Scheme for COV-ID on saliva spiked with viral and RNA and absorbed on paper. ( D ) Same as ( B ) but on saliva absorbed on paper. ( E ) SARS-CoV-2 virus was added to saliva and prepared as in ( C ).Reverse transcription loop-mediated isothermal amplification (RT-LAMP) and sequencing was carried out in presence of calibration standard RNA. Viral reads are presented as ratio against the sum of STATH and N2 synthetic calibration standard (SCS) reads. Positive threshold was set as 2x maximum value in negative saliva and indicated by dashed horizontal line. (F–G) Paper-based COV-ID workflow ( F ) and cost calculations ( G ). Saliva is collected orally on a precut strip of paper, from which a 2 mm square would be cut out and added to a reaction vessel containing TCEP/EDTA inactivation buffer and processed as shown in ( C ). Uncropped blot for Figure 4B . Uncropped blot for Figure 4D .

    Journal: eLife

    Article Title: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    doi: 10.7554/eLife.69949

    Figure Lengend Snippet: COV-ID on saliva collected on paper. ( A ) Scheme for COV-ID on viral RNA absorbed on paper. ( B ) PCR reactions from paper samples immersed in water with indicated viral concentrations then amplified with N2 COV-ID primers. ( C ) Scheme for COV-ID on saliva spiked with viral and RNA and absorbed on paper. ( D ) Same as ( B ) but on saliva absorbed on paper. ( E ) SARS-CoV-2 virus was added to saliva and prepared as in ( C ).Reverse transcription loop-mediated isothermal amplification (RT-LAMP) and sequencing was carried out in presence of calibration standard RNA. Viral reads are presented as ratio against the sum of STATH and N2 synthetic calibration standard (SCS) reads. Positive threshold was set as 2x maximum value in negative saliva and indicated by dashed horizontal line. (F–G) Paper-based COV-ID workflow ( F ) and cost calculations ( G ). Saliva is collected orally on a precut strip of paper, from which a 2 mm square would be cut out and added to a reaction vessel containing TCEP/EDTA inactivation buffer and processed as shown in ( C ). Uncropped blot for Figure 4B . Uncropped blot for Figure 4D .

    Article Snippet: To prepare the in vitro transcription template for SARS-CoV-2 N2 synthetic calibration standard RNA, we performed RT-PCR using Power SYBR RNA-to-Ct kit (Thermo Cat. 4389986) of heat inactivated SARS-CoV-2 (BEI Resources Cat. NR-52286) using the following primers: N2-B3 and N2-spike-T7 S. PCR product was purified and used as a template for in vitro transcription using HiScribe T7 transcription kit (NEB Cat. E2040S).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Stripping Membranes

    Analysis of SP6 promoter sequences. a 5 ′ RACE-seq using SP6 polymerase. Normalized average nucleotide composition from position +2 to +16 in RNA transcribed by SP6 RNA polymerase from a randomized SP6 promoter library. Substantial sequence preference was observed until the +3 nucleotide position. b Box plot showing relative abundances of +2 to +16 SP6 promoter variants detected in 5 ′ RACE-seq, separated by +2/3 dinucleotide sequence. All variants have a G at +1. Promoters with +1 to +3 GAA showed highest activity. Each whisker plot represents 948–985 +1 to +8 motifs, dependent on homopolymer filtering. Whiskers reach to 1.5× IQR away from the 1st/3rd quartile. c IVT using high ranking +2 to +8 SP6 promoter variants with the indicated +2/3 dinucleotides. The +2/3 dinucleotide sequence appeared as main determinant of SP6 transcriptional activity. d IVT using SP6 promoter templates harboring +1 to +3 GAA followed by +4 to +8 sequence motifs of varying 5 ′ RACE-seq rank. IVT was performed for 2 h. Shown is the resulting fold amplification of the template DNA. Sequence elements after +4 showed no effects on SP6 promoter activity. All error bars represent standard deviation for triplicate experiments.

    Journal: Communications Biology

    Article Title: Maximizing transcription of nucleic acids with efficient T7 promoters

    doi: 10.1038/s42003-020-01167-x

    Figure Lengend Snippet: Analysis of SP6 promoter sequences. a 5 ′ RACE-seq using SP6 polymerase. Normalized average nucleotide composition from position +2 to +16 in RNA transcribed by SP6 RNA polymerase from a randomized SP6 promoter library. Substantial sequence preference was observed until the +3 nucleotide position. b Box plot showing relative abundances of +2 to +16 SP6 promoter variants detected in 5 ′ RACE-seq, separated by +2/3 dinucleotide sequence. All variants have a G at +1. Promoters with +1 to +3 GAA showed highest activity. Each whisker plot represents 948–985 +1 to +8 motifs, dependent on homopolymer filtering. Whiskers reach to 1.5× IQR away from the 1st/3rd quartile. c IVT using high ranking +2 to +8 SP6 promoter variants with the indicated +2/3 dinucleotides. The +2/3 dinucleotide sequence appeared as main determinant of SP6 transcriptional activity. d IVT using SP6 promoter templates harboring +1 to +3 GAA followed by +4 to +8 sequence motifs of varying 5 ′ RACE-seq rank. IVT was performed for 2 h. Shown is the resulting fold amplification of the template DNA. Sequence elements after +4 showed no effects on SP6 promoter activity. All error bars represent standard deviation for triplicate experiments.

    Article Snippet: Generation of 5′ RACE-seq libraries Totally, 10 ng of dsDNA template (T7_15N/SP6_15N) containing a T7/SP6 RNA polymerase promoter randomized from position +2 to +16 (gBlocks Gene Fragments, Integrated DNA Technologies) was used as input for IVT in a 20 µl reaction using 1.5 µl T7 or T6 RNA polymerase mix and 7.5 mM each of GTP, ATP, CTP, and UTP from the HiScribe T7/SP6 RNA Synthesis Kit (NEB E2040S, E2070S).

    Techniques: Sequencing, Activity Assay, Whisker Assay, Amplification, Standard Deviation