The Experts below are selected from a list of 93 Experts worldwide ranked by ideXlab platform
René F. Ketting - One of the best experts on this subject based on the ideXlab platform.
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RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.
MethodsX, 2019Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.
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RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs
2018Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans . Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans , maintain the 5′ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli . Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.
Miguel Vasconcelos Almeida - One of the best experts on this subject based on the ideXlab platform.
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RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.
MethodsX, 2019Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.
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RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs
2018Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans . Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans , maintain the 5′ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli . Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.
António Miguel De Jesus Domingues - One of the best experts on this subject based on the ideXlab platform.
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RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.
MethodsX, 2019Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.
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RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs
2018Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans . Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans , maintain the 5′ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli . Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.
Hanna Lukas - One of the best experts on this subject based on the ideXlab platform.
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RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.
MethodsX, 2019Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.
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RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs
2018Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans . Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans , maintain the 5′ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli . Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.
Maria Mendez-lago - One of the best experts on this subject based on the ideXlab platform.
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RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.
MethodsX, 2019Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.
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RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs
2018Co-Authors: Miguel Vasconcelos Almeida, António Miguel De Jesus Domingues, Hanna Lukas, Maria Mendez-lago, René F. KettingAbstract:RNA interference was first described in the nematode Caenorhabditis elegans . Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans , maintain the 5′ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid Pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available Pyrophosphatase isolated from E. coli . Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA Pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.