t4 rna ligase 2 New England Biolabs Search Results


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    New England Biolabs t4 rna ligase 2
    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated <t>T4</t> RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.
    T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2023 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs kq t4 rna ligase 2
    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, <t>Rnl2:</t> <t>T4</t> RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA <t>ligase</t> 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.
    Kq T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 rna ligase 2 k227q
    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, <t>Rnl2:</t> <t>T4</t> RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA <t>ligase</t> 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.
    T4 Rna Ligase 2 K227q, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 rnl2 reaction buffer
    Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.
    T4 Rnl2 Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Journal: Cell

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    doi: 10.1016/j.cell.2018.07.022

    Figure Lengend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Article Snippet: The reactions were carried out in 20 μL with 1x T4 RNA ligase 2 truncated buffer (NEB) supplemented with PEG-8000 at 10% final concentration, 0.25 U/μl RiboLock inhibitor (Thermo Fisher Scientific), 3 pmol of the 5′ FAM-labeled 44-mer oligonucleotide RNA44 (Future Synthesis) and 300 U T4 RNA ligase 2 truncated (NEB) for 18h at 18°C.

    Techniques: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.

    Journal: bioRxiv

    Article Title: Multiple ribonuclease A family members cleave transfer RNAs in response to stress

    doi: 10.1101/811174

    Figure Lengend Snippet: Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.

    Article Snippet: For RNA ligase 2-based ligation reaction, the annealed samples were incubated with 5 µl of 1x reaction buffer, 5 U (0.5 µl) of T4 RNA ligase 2 (Rnl2) (New England Biolabs) and 40 U (1 µl) of RNasin (Promega) at 37°C for 1 hr, followed by overnight incubation at 4°C.

    Techniques: Ligation, Staining, Northern Blot

    Effect of PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Effect of PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Activity Assay, Labeling, Ligation, Binding Assay

    Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Activity Assay, Incubation, Staining, Electrophoretic Mobility Shift Assay, Binding Assay

    Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Incubation, Staining, Ligation, Binding Assay

    Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Ligation, Labeling, Incubation, Acrylamide Gel Assay, Nucleic Acid Electrophoresis, Binding Assay

    Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Ligation, Incubation, Staining, Binding Assay

    Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments. * denotes difference in means p

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments. * denotes difference in means p

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Purification, Activity Assay, Staining, Marker, Incubation, Labeling, Binding Assay

    Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Activity Assay, Labeling, Ligation, Binding Assay

    Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Journal: BMC Biotechnology

    Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

    doi: 10.1186/1472-6750-11-72

    Figure Lengend Snippet: Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.

    Article Snippet: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins Mutations were introduced into the T4 Rnl2tr coding sequence by site-directed mutagenesis using the Phusion Site-directed Mutagenesis Kit (New England Biolabs).

    Techniques: Labeling, Ligation, Binding Assay

    Comparison of T4 DNA Ligase, T4 RNA Ligase 2 and SplintR Ligase for their ability to join different length DNA probes hybridized to miR-122. ( A ) Pairs of DNA oligonucleotides complementary to miR-122 were used to determine the minimum DNA:RNA overlap required for ligation. The miR-122 sequence (red) is complementary to probe A (green) and probe B (black). Probe A and probe B are designed to scan the miRNA sequence in two base increments. Probe A has a 5′ FAM label and probe B has a 5′ phosphate that allows ligation. Each pair of DNA probes is named by the number of nucleotides (nt) that are complementary to the 22 nt in miR-122, for example probe A8 has 8 nt and B14 has 14 nt complementary to miR-122. ( B ) The ligation of FAM labeled probe A to probe B is visualized by a fluorescent scan after electrophoresis on a 15% TBE urea acrylamide gel. Probe B is not observed because only probe A has a FAM label. Since there is more probe A than miR-122 each reaction has both ligated and unligated probe. Lane M, at the left of the gel, denotes the size in nucleotides of the FAM labeled ssDNA marker. The specific components and reaction conditions are described in Materials and Methods. ( C ) The ligation of DNA probes hybridized to miR-122 by SplintR Ligase, T4 DNA Ligase or T4 RNA Ligase 2 (T4 RL2) (indicated on the left) at different incubation times was measured by capillary electrophoresis (CE) fragment analysis. The traces are shown for 5, 30 and 60 minute ligations of probes gA10 and gB12 hybridized to miR-122. Position of the 32 nt substrate (32(S)) and the 65 nt ligated product (65(P)) are indicated on the top of the panels. Reaction conditions are listed in Materials and Methods. ( D ) Ligation time course for the three enzymes, SpllintR (red), T4 DNA ligase (black) and T4 RNA ligase 2 (green) was measured by CE. The percent of ligation is indicated on the left of the graph.

    Journal: Nucleic Acids Research

    Article Title: Sensitive and specific miRNA detection method using SplintR Ligase

    doi: 10.1093/nar/gkw399

    Figure Lengend Snippet: Comparison of T4 DNA Ligase, T4 RNA Ligase 2 and SplintR Ligase for their ability to join different length DNA probes hybridized to miR-122. ( A ) Pairs of DNA oligonucleotides complementary to miR-122 were used to determine the minimum DNA:RNA overlap required for ligation. The miR-122 sequence (red) is complementary to probe A (green) and probe B (black). Probe A and probe B are designed to scan the miRNA sequence in two base increments. Probe A has a 5′ FAM label and probe B has a 5′ phosphate that allows ligation. Each pair of DNA probes is named by the number of nucleotides (nt) that are complementary to the 22 nt in miR-122, for example probe A8 has 8 nt and B14 has 14 nt complementary to miR-122. ( B ) The ligation of FAM labeled probe A to probe B is visualized by a fluorescent scan after electrophoresis on a 15% TBE urea acrylamide gel. Probe B is not observed because only probe A has a FAM label. Since there is more probe A than miR-122 each reaction has both ligated and unligated probe. Lane M, at the left of the gel, denotes the size in nucleotides of the FAM labeled ssDNA marker. The specific components and reaction conditions are described in Materials and Methods. ( C ) The ligation of DNA probes hybridized to miR-122 by SplintR Ligase, T4 DNA Ligase or T4 RNA Ligase 2 (T4 RL2) (indicated on the left) at different incubation times was measured by capillary electrophoresis (CE) fragment analysis. The traces are shown for 5, 30 and 60 minute ligations of probes gA10 and gB12 hybridized to miR-122. Position of the 32 nt substrate (32(S)) and the 65 nt ligated product (65(P)) are indicated on the top of the panels. Reaction conditions are listed in Materials and Methods. ( D ) Ligation time course for the three enzymes, SpllintR (red), T4 DNA ligase (black) and T4 RNA ligase 2 (green) was measured by CE. The percent of ligation is indicated on the left of the graph.

    Article Snippet: T4 RNA Ligase 2 reactions were performed in 1X RNA Ligase 2 buffer (NEB).

    Techniques: Ligation, Sequencing, Labeling, Electrophoresis, Acrylamide Gel Assay, Marker, Incubation

    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Journal: Cell

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    doi: 10.1016/j.cell.2018.07.022

    Figure Lengend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Article Snippet: The reactions were carried out in 20 μL with 1x T4 RNA ligase 2 truncated buffer (NEB) supplemented with PEG-8000 at 10% final concentration, 0.25 U/μl RiboLock inhibitor (Thermo Fisher Scientific), 3 pmol of the 5′ FAM-labeled 44-mer oligonucleotide RNA44 (Future Synthesis) and 300 U T4 RNA ligase 2 truncated (NEB) for 18h at 18°C.

    Techniques: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    Sequencing cDNA generated from N21 RNA libraries . a Number of reads for the 100 most abundant sequences in the N21 libraries, prepared with Illumina ( red ) or HD adapters ( blue ). b-d Frequencies of predicted nucleotide base-pairing per position for N21 insert ( b ), N21 insert and 3’ adapter ( c ) and 5’ adapter, insert and 3’ adapter ( d ). In ( c ) and ( d ) vertical dotted line indicates ligation point. Red line denotes data obtained with Illumina protocol, blue line with HD protocol and grey line randomly generated sets of 21nt sequences. Bars indicate minimum and maximum values in all replicates. Horizontal bars at bottom indicate sequence region: green , insert; red , 3’ adapter; blue , 5’ adapter. For insert folding frequencies obtained with random sequences are more closely matched by HD data (R 2 = 0.83) than by Illumina data (R 2 = 0.60). e Comparison of T4 Rnl2 ligase activity on substrates with ss flaps of differing nucleotide lengths upstream or downstream of ligation site. In vitro ligation assay of RNA-DNA duplexes with either a nick (0NT) or ss flaps up- or downstream from the ligation site was carried out at 25°C for 30 min. Substrates with ss flaps > 2nt in length upstream from the ligation site are inefficiently ligated. The diagram illustrates the position of the flaps, the fluorescein reporter group ( star ) and the backbone oligonucleotide ( black ). If ligation occurs the size of the nucleic acid attached to the fluorescein increases as visualised by 15% PAGE.

    Journal: Silence

    Article Title: Reducing ligation bias of small RNAs in libraries for next generation sequencing

    doi: 10.1186/1758-907X-3-4

    Figure Lengend Snippet: Sequencing cDNA generated from N21 RNA libraries . a Number of reads for the 100 most abundant sequences in the N21 libraries, prepared with Illumina ( red ) or HD adapters ( blue ). b-d Frequencies of predicted nucleotide base-pairing per position for N21 insert ( b ), N21 insert and 3’ adapter ( c ) and 5’ adapter, insert and 3’ adapter ( d ). In ( c ) and ( d ) vertical dotted line indicates ligation point. Red line denotes data obtained with Illumina protocol, blue line with HD protocol and grey line randomly generated sets of 21nt sequences. Bars indicate minimum and maximum values in all replicates. Horizontal bars at bottom indicate sequence region: green , insert; red , 3’ adapter; blue , 5’ adapter. For insert folding frequencies obtained with random sequences are more closely matched by HD data (R 2 = 0.83) than by Illumina data (R 2 = 0.60). e Comparison of T4 Rnl2 ligase activity on substrates with ss flaps of differing nucleotide lengths upstream or downstream of ligation site. In vitro ligation assay of RNA-DNA duplexes with either a nick (0NT) or ss flaps up- or downstream from the ligation site was carried out at 25°C for 30 min. Substrates with ss flaps > 2nt in length upstream from the ligation site are inefficiently ligated. The diagram illustrates the position of the flaps, the fluorescein reporter group ( star ) and the backbone oligonucleotide ( black ). If ligation occurs the size of the nucleic acid attached to the fluorescein increases as visualised by 15% PAGE.

    Article Snippet: Approximately 200 ng of a small RNA-enriched sample was ligated to the pre-adenylated 3' adapter (custom synthesised by Bioo Scientific) with T4 Rnl2 truncated ligase (NEB).

    Techniques: Sequencing, Generated, Ligation, Activity Assay, In Vitro, Polyacrylamide Gel Electrophoresis

    Scheme depicting the experimental approach and HD adapters . a Data were generated to analyse the sequence preferences of T4 Rnl1 and T4 Rnl2 using a degenerate RNA library (N21 RNA). b HD adapters include degenerate tags at the end of the adapters that allow the formation of stable secondary structures for more sequences and reduce RNA ligase-dependent sequence bias. Panel ( c ) shows the structure of miR-29b with the Illumina adapters ( top ) and some of the structures formed by HD adapters ( bottom ). We found 1,031 distinct structures originating from 12,479 tag combinations.

    Journal: Silence

    Article Title: Reducing ligation bias of small RNAs in libraries for next generation sequencing

    doi: 10.1186/1758-907X-3-4

    Figure Lengend Snippet: Scheme depicting the experimental approach and HD adapters . a Data were generated to analyse the sequence preferences of T4 Rnl1 and T4 Rnl2 using a degenerate RNA library (N21 RNA). b HD adapters include degenerate tags at the end of the adapters that allow the formation of stable secondary structures for more sequences and reduce RNA ligase-dependent sequence bias. Panel ( c ) shows the structure of miR-29b with the Illumina adapters ( top ) and some of the structures formed by HD adapters ( bottom ). We found 1,031 distinct structures originating from 12,479 tag combinations.

    Article Snippet: Approximately 200 ng of a small RNA-enriched sample was ligated to the pre-adenylated 3' adapter (custom synthesised by Bioo Scientific) with T4 Rnl2 truncated ligase (NEB).

    Techniques: Generated, Sequencing

    Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.

    Journal: Nucleic Acids Research

    Article Title: Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

    doi: 10.1093/nar/gkx073

    Figure Lengend Snippet: Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.

    Article Snippet: The ligation reaction was performed in 20 μl containing 1× T4 Rnl2 reaction buffer (NEB), 12.5% (w/v) polyethylene glycol (PEG) 8000 or PEG 4000 and 5 units of T4 RNA Ligase 2 (NEB).

    Techniques: Ligation, Transformation Assay