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  • 97
    InGex tgirt iii
    <t>TGIRT-seq</t> reads map mostly to protein-coding genes but with greater representation of small ncRNAs than TruSeq libraries. ( A ) for different library preparation methods for numbered replicates of Samples A–D. ( B ) Stacked bar graphs showing the percentage of small noncoding RNA reads that map to different classes of small ncRNAs for different library preparation methods for numbered replicates of Samples A–D. MiscRNA includes ribozymes, such as RNase P RNA, imprinted transcripts, such as Xist, and other transcripts that cannot be classified into other RNA annotation categories. ( Left panels) TGIRT-seq; ( middle panels) TruSeq v2 (from ABRF at <t>three</t> different sites, L/R/V); ( right panels) TruSeq v3 (from ABRF at site W). Features and small ncRNA classes are color coded as indicated to the right of the bar graphs.
    Tgirt Iii, supplied by InGex, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InGex tgirt iii enzyme
    <t>TGIRT</t> ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the <t>TGIRT-III</t> enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.
    Tgirt Iii Enzyme, supplied by InGex, used in various techniques. Bioz Stars score: 95/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgirt iii enzyme/product/InGex
    Average 95 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    tgirt iii enzyme - by Bioz Stars, 2020-04
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    93
    InGex tgirt iii reverse transcriptase
    <t>TGIRT</t> ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the <t>TGIRT-III</t> enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.
    Tgirt Iii Reverse Transcriptase, supplied by InGex, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgirt iii reverse transcriptase/product/InGex
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    tgirt iii reverse transcriptase - by Bioz Stars, 2020-04
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    93
    InGex tgirt iii group ii intron rt
    <t>TGIRT</t> ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the <t>TGIRT-III</t> enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.
    Tgirt Iii Group Ii Intron Rt, supplied by InGex, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgirt iii group ii intron rt/product/InGex
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    TGIRT-seq reads map mostly to protein-coding genes but with greater representation of small ncRNAs than TruSeq libraries. ( A ) for different library preparation methods for numbered replicates of Samples A–D. ( B ) Stacked bar graphs showing the percentage of small noncoding RNA reads that map to different classes of small ncRNAs for different library preparation methods for numbered replicates of Samples A–D. MiscRNA includes ribozymes, such as RNase P RNA, imprinted transcripts, such as Xist, and other transcripts that cannot be classified into other RNA annotation categories. ( Left panels) TGIRT-seq; ( middle panels) TruSeq v2 (from ABRF at three different sites, L/R/V); ( right panels) TruSeq v3 (from ABRF at site W). Features and small ncRNA classes are color coded as indicated to the right of the bar graphs.

    Journal: RNA

    Article Title: RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase

    doi: 10.1261/rna.055558.115

    Figure Lengend Snippet: TGIRT-seq reads map mostly to protein-coding genes but with greater representation of small ncRNAs than TruSeq libraries. ( A ) for different library preparation methods for numbered replicates of Samples A–D. ( B ) Stacked bar graphs showing the percentage of small noncoding RNA reads that map to different classes of small ncRNAs for different library preparation methods for numbered replicates of Samples A–D. MiscRNA includes ribozymes, such as RNase P RNA, imprinted transcripts, such as Xist, and other transcripts that cannot be classified into other RNA annotation categories. ( Left panels) TGIRT-seq; ( middle panels) TruSeq v2 (from ABRF at three different sites, L/R/V); ( right panels) TruSeq v3 (from ABRF at site W). Features and small ncRNA classes are color coded as indicated to the right of the bar graphs.

    Article Snippet: Half of the recovered RNA was used for cDNA synthesis via TGIRT template switching with 1 μM TGIRT-III RT (InGex, LLC) for 15 min at 60°C, as previously described ( ).

    Techniques:

    TGIRT ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the TGIRT-III enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.

    Journal: Scientific Reports

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

    doi: 10.1038/s41598-017-09064-w

    Figure Lengend Snippet: TGIRT ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the TGIRT-III enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.

    Article Snippet: Reactions were done with 5–50 ng DNA substrate, 100 nM annealed template-primer substrate, and TGIRT-III enzyme (400 units, 1 mM; InGex, LLC; St. Louis) in 20 μl of reaction medium containing 420 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl, pH 7.5, 5 mM DTT and 1 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP).

    Techniques: DNA Sequencing, DNA Synthesis, Blocking Assay, DNA Ligation, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Multiplexing

    Bisulfite sequencing of plasma cfDNA. Three separate TGIRT-seq libraries were each constructed from ~5 ng of bisulfite-treated plasma DNA from a healthy male individual, and sequenced to obtain TGIRT-seq datasets BPD1-3 (see Supplementary Table S4 ). The datasets were combined and analyzed to identify DNA methylation sites, as described in Methods. ( a ) Annotation of DNA methylation sites in the human genome and determination of DNA methylation densities by TGIRT-seq of bisulfite-treated plasma cfDNA. Tracks from the inner to the outer circle represent: (1) annotations of Type II biomarkers that are highly variable in methylation density across tissues; (2) annotations of Type I biomarkers that are highly tissue specific (color-coded); (3) bar graphs of methylation densities within the annotated regions based on TGIRT-seq of bisulfite-treated plasma cfDNA; (4) genome coordinates. ( b ) Tissues-of-origin of plasma cfDNA from a healthy male individual determined by TGIRT-seq of bisulfite-treated DNA. The pie chart shows the percent contributions of different tissues in combined datasets PB1-3 determined by quadratic programming 7 . Neutrophils are derived from myeloid precursors, and lymphocytes are a combination of T-cells and B-cells. Comparison of tissues-of-origin of plasma cfDNA determined as in panel ( b ) from the three independent datasets BPD1-3 are shown in Supplementary Fig. 11 . Pearson’s correlation coefficients were ≥0.96 for each pairwise combination of the three individual datasets.

    Journal: Scientific Reports

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

    doi: 10.1038/s41598-017-09064-w

    Figure Lengend Snippet: Bisulfite sequencing of plasma cfDNA. Three separate TGIRT-seq libraries were each constructed from ~5 ng of bisulfite-treated plasma DNA from a healthy male individual, and sequenced to obtain TGIRT-seq datasets BPD1-3 (see Supplementary Table S4 ). The datasets were combined and analyzed to identify DNA methylation sites, as described in Methods. ( a ) Annotation of DNA methylation sites in the human genome and determination of DNA methylation densities by TGIRT-seq of bisulfite-treated plasma cfDNA. Tracks from the inner to the outer circle represent: (1) annotations of Type II biomarkers that are highly variable in methylation density across tissues; (2) annotations of Type I biomarkers that are highly tissue specific (color-coded); (3) bar graphs of methylation densities within the annotated regions based on TGIRT-seq of bisulfite-treated plasma cfDNA; (4) genome coordinates. ( b ) Tissues-of-origin of plasma cfDNA from a healthy male individual determined by TGIRT-seq of bisulfite-treated DNA. The pie chart shows the percent contributions of different tissues in combined datasets PB1-3 determined by quadratic programming 7 . Neutrophils are derived from myeloid precursors, and lymphocytes are a combination of T-cells and B-cells. Comparison of tissues-of-origin of plasma cfDNA determined as in panel ( b ) from the three independent datasets BPD1-3 are shown in Supplementary Fig. 11 . Pearson’s correlation coefficients were ≥0.96 for each pairwise combination of the three individual datasets.

    Article Snippet: Reactions were done with 5–50 ng DNA substrate, 100 nM annealed template-primer substrate, and TGIRT-III enzyme (400 units, 1 mM; InGex, LLC; St. Louis) in 20 μl of reaction medium containing 420 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl, pH 7.5, 5 mM DTT and 1 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP).

    Techniques: Methylation Sequencing, Construct, DNA Methylation Assay, Methylation, Derivative Assay