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    Thermostable 5 App DNA RNA Ligase
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    Thermostable 5 App DNA RNA Ligase 50 rxns
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    Thermostable 5 App DNA RNA Ligase
    Thermostable 5 App DNA RNA Ligase 50 rxns
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    1) Product Images from "Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction"

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-44457-z

    Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.
    Figure Legend Snippet: Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.

    Techniques Used: Construct, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.
    Figure Legend Snippet: TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.

    Techniques Used: Sequencing, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Modification, Chromatin Immunoprecipitation

    TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.
    Figure Legend Snippet: TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.

    Techniques Used:

    2) Product Images from "Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction"

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-44457-z

    Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.
    Figure Legend Snippet: Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.

    Techniques Used: Construct, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.
    Figure Legend Snippet: TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.

    Techniques Used: Sequencing, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Modification, Chromatin Immunoprecipitation

    TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.
    Figure Legend Snippet: TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.

    Techniques Used:

    3) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.
    Figure Legend Snippet: Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.

    Techniques Used: Modification, Ligation

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    4) Product Images from "Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq"

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011337

    Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.
    Figure Legend Snippet: Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Techniques Used:

    5) Product Images from "Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09064-w

    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.
    Figure Legend 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.

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

    6) Product Images from "Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09064-w

    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.
    Figure Legend 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.

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

    7) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.
    Figure Legend Snippet: Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.

    Techniques Used: Modification, Ligation

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    8) Product Images from "Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq"

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011337

    Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.
    Figure Legend Snippet: Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Techniques Used:

    Related Articles

    Flow Cytometry:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Amplification:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    DNA Synthesis:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    DNA Ligation:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    Ligation:

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: .. These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ). ..

    Mutagenesis:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum . ..

    Isolation:

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: .. Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum . ..

    Introduce:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Sequencing:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
    Article Snippet: .. These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ). ..

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    DNA Sequencing:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Polymerase Chain Reaction:

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After the completion of DNA synthesis, a second DNA-seq adapter containing a reverse complement of an Illumina read 1 adapter sequence (R1R) is ligated to the 3′ end of the DNA product strand by an efficient single-stranded DNA ligation using thermostable 5′ AppDNA/RNA ligase (New England Biolabs), and finally the target DNA sequences are amplified by PCR using primers that introduce Illumina flow cell capture sites and sample barcodes for sequencing. .. An unique molecular identifier (UMI), a randomized nucleotide sequence that tags individual DNA products, can be incorporated into either the 5′ or 3′ adapter (shown in Fig. for the R1R adapter, the location of the UMI in the present work).

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
    Article Snippet: .. After cleaning up by using a Nucleospin Gel and PCR cleanup kit (Clontech), a 5′App/3′-CpSp blocked R1R adapter with a 13-nt UMI at its 5′ end (denoted R1R-UMI) was ligated to the 3′ end of the cDNA by using Thermostable 5′ AppDNA/RNA Ligase as specified by manufacturer’s protocol (New England Biolabs). .. The ligated products were cleaned up by Nucleospin Gel and PCR cleanup kit (Clontech) and PCR amplified by using a KAPA Library Amplification Kit (KAPA) with 500 nM each of Illumina multiplex and barcode primers, with the 5′ primer adding a P5 capture site and the 3′ primer adding a sequencing barcode and P7 capture site (see Supplementary Table ).

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

    RNA Sequencing Assay:

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
    Article Snippet: .. After reverse transcription, a second RNA-seq adapter (R1R DNA; containing the reverse complement of an Illumina Read 1 sequence) is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. .. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).

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    New England Biolabs app dna rna ligase
    Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized <t>RNA</t> oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity <t>DNA</t> chip. M: internal markers.
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    Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.

    Journal: Scientific Reports

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    doi: 10.1038/s41598-019-44457-z

    Figure Lengend Snippet: Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. 1A . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.

    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.

    Techniques: Construct, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.

    Journal: Scientific Reports

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    doi: 10.1038/s41598-019-44457-z

    Figure Lengend Snippet: TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.

    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.

    Techniques: Sequencing, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Modification, Chromatin Immunoprecipitation

    TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.

    Journal: Scientific Reports

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    doi: 10.1038/s41598-019-44457-z

    Figure Lengend Snippet: TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3′-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3′-overhang nucleotides. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.

    Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.

    Techniques:

    Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.

    Journal: PLoS ONE

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    doi: 10.1371/journal.pone.0094619

    Figure Lengend Snippet: Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs. The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.

    Article Snippet: Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum .

    Techniques: Modification, Ligation

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Journal: PLoS ONE

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    doi: 10.1371/journal.pone.0094619

    Figure Lengend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Article Snippet: Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Methanobacterium thermoautotrophicum .

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Journal: The Journal of Biological Chemistry

    Article Title: Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

    doi: 10.1074/jbc.RA119.011337

    Figure Lengend Snippet: Models of template switching and non-templated nucleotide addition reactions. A , template switching to an acceptor RNA from an RNA template/DNA primer heteroduplex with a 1-nt 3′ overhang added by NTA after completion of cDNA synthesis or as an artificial starter duplex. B , NTA to a blunt-end RNA/DNA heteroduplex in the absence of an acceptor nucleic acid. C , template-switching to an acceptor RNA from a blunt-end RNA/DNA heteroduplex without NTA. See under “Discussion” for details.

    Article Snippet: These 3′ biases, which are restricted to the 3′-terminal nucleotide of the acceptor, account for about half of the sequence bias in TGIRT-seq, the remainder coming from the Thermostable 5′ App RNA/DNA Ligase (New England Biolabs) used for R1R adapter ligation (see A ) ( ).

    Techniques: