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nebnext small rna sequencing kit  (New England Biolabs)


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    New England Biolabs nebnext small rna sequencing kit
    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    1) Product Images from "NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes"

    Article Title: NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkag027

    NUP98 interacts with an RNA motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding sequence; GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
    Figure Legend Snippet: NUP98 interacts with an RNA motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding sequence; GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.

    Techniques Used: Transfection, Infection, Immunoprecipitation, Control, Comparison, Focus Forming Assay, CRISPR, RNA Binding Assay, Sequencing, Purification, Construct, Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Concentration Assay



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    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    NUP98 interacts with an <t>RNA</t> motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding <t>sequence;</t> GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.
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    NUP98 interacts with an RNA motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding sequence; GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.

    Journal: Nucleic Acids Research

    Article Title: NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes

    doi: 10.1093/nar/gkag027

    Figure Lengend Snippet: NUP98 interacts with an RNA motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding sequence; GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.

    Article Snippet: The RNA was then extracted and used to prepare a cDNA library using the NEBNext small RNA sequencing kit (New England Biolabs) according to manufacturer’s instructions.

    Techniques: Transfection, Infection, Immunoprecipitation, Control, Comparison, Focus Forming Assay, CRISPR, RNA Binding Assay, Sequencing, Purification, Construct, Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Concentration Assay