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    New England Biolabs dtb gtp
    Dtb Gtp, 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
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    New England Biolabs dtb gtp
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    3 desthiobiotin gtp dtb gtp  (New England Biolabs)


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    New England Biolabs 3 desthiobiotin gtp dtb gtp
    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog <t>(3′-desthiobiotin-GTP)</t> using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    3 Desthiobiotin Gtp Dtb Gtp, 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
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    3 desthiobiotin gtp dtb gtp - by Bioz Stars, 2024-06
    94/100 stars

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    1) Product Images from "Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq"

    Article Title: Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

    Journal: Genome Research

    doi: 10.1101/gr.275784.121

    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    Figure Legend Snippet: ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Techniques Used: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro

    3 desthiobiotin gtp dtb gtp  (New England Biolabs)


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    New England Biolabs 3 desthiobiotin gtp dtb gtp
    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog <t>(3′-desthiobiotin-GTP)</t> using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    3 Desthiobiotin Gtp Dtb Gtp, 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/3 desthiobiotin gtp dtb gtp/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    3 desthiobiotin gtp dtb gtp - by Bioz Stars, 2024-06
    94/100 stars

    Images

    1) Product Images from "Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq"

    Article Title: Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

    Journal: Genome Research

    doi: 10.1101/gr.275784.121

    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    Figure Legend Snippet: ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Techniques Used: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro

    desthiobiotin gtp dtb gtp  (New England Biolabs)


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    New England Biolabs desthiobiotin gtp dtb gtp
    ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified <t>GTP</t> analog <t>(3’-desthiobiotin-GTP)</t> using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    Desthiobiotin Gtp Dtb Gtp, 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/desthiobiotin gtp dtb gtp/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    desthiobiotin gtp dtb gtp - by Bioz Stars, 2024-06
    94/100 stars

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    1) Product Images from "ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases"

    Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

    Journal: bioRxiv

    doi: 10.1101/696559

    ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    Figure Legend Snippet: ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Techniques Used: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro, RNA Sequencing Assay

    dtb gtp  (New England Biolabs)


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    New England Biolabs dtb gtp
    Dtb Gtp, 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
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    https://www.bioz.com/result/dtb gtp/product/New England Biolabs
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    New England Biolabs 3 desthiobiotin gtp dtb gtp
    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog <t>(3′-desthiobiotin-GTP)</t> using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
    3 Desthiobiotin Gtp Dtb Gtp, 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/3 desthiobiotin gtp dtb gtp/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
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    New England Biolabs desthiobiotin gtp dtb gtp
    ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified <t>GTP</t> analog <t>(3’-desthiobiotin-GTP)</t> using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).
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    ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Journal: Genome Research

    Article Title: Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

    doi: 10.1101/gr.275784.121

    Figure Lengend Snippet: ReCappable-seq. (A) Principle of ReCappable-seq. (1) RNA is subjected to decapping with yDcpS, which acts on capped transcripts originating from Pol II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3′-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. (2) Differentiation of Pol II from the non–Pol II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP before the yDcpS treatment, in order to remove the 5′ triphosphate from non–Pol II transcripts. (B) RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as RNA18S1 rRNA as an example of a processed transcript; ACTB, RPL19, MALAT1, FKBP5, TMSB10, H3C10, and HIST2H3B as examples of capped transcripts; RMRP, RPPH1, and RN7SK as examples of Pol III transcripts (with RN7SK having a 5′ methylated triphosphate and therefore being resistant to CIP treatment; see main text); and ERCC190 and FLUC as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5′ end. The Cq values are available in Supplemental Figure S1F. (C) Example of a Pol II TSS in the TMSB10 locus: The same positions (shaded in pink) are found in the CAGE data set. CIP treatment intensifies the signal, consistent with a Pol II TSS. (D) Example of Pol III TSS corresponding to the start of two vault RNAs (vtRNA1-1 and vtRNA1-2) located on Chr 5. The positions (shaded in pink) are missing in the CAGE data set. CIP treatment reduces the signal, consistent with non–Pol II TSS. In C and D, the tracks correspond to ReCappable-seq, CIP-ReCappable-seq, and CAGE read coverage (number of reads). All libraries were down-sampled to the same number of total mapped reads (63,300,000) to facilitate comparison. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Article Snippet: Capping with 3′ desthiobiotin GTP (DTB-GTP) was performed in 50 µL total volume with 5 µL VCE (NEB M02080) and 0.5 mM DTB-GTP (NEB N0761), in the absence of SAM for 40 min at 37°C.

    Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro

    ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Journal: bioRxiv

    Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

    doi: 10.1101/696559

    Figure Lengend Snippet: ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Article Snippet: Capping with 3’ Desthiobiotin GTP (DTB-GTP) was performed in 50 µl total volume with 5 µL Vaccinia capping enzyme (NEB M02080) and 0.5 mM DTB-GTP (NEB N0761), in the absence of SAM for 40 min at 37°C.

    Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro, RNA Sequencing Assay