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  • 99
    New England Biolabs t4 rnl2 truncated
    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 <t>T4</t> 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.
    T4 Rnl2 Truncated, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rnl2 truncated/product/New England Biolabs
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    t4 rnl2 truncated - by Bioz Stars, 2020-07
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    99
    New England Biolabs t4 rnl2 truncated reaction buffer
    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 <t>T4</t> 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.
    T4 Rnl2 Truncated Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rnl2 truncated reaction buffer/product/New England Biolabs
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    t4 rnl2 truncated reaction buffer - by Bioz Stars, 2020-07
    99/100 stars
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    99
    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 2038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 2/product/New England Biolabs
    Average 99 stars, based on 2038 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 - by Bioz Stars, 2020-07
    99/100 stars
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    99
    New England Biolabs t4 rna ligase 2 k227q
    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. <t>T4</t> RNA <t>Ligase</t> 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 <t>K227Q</t> (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.
    T4 Rna Ligase 2 K227q, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 2 k227q/product/New England Biolabs
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 k227q - by Bioz Stars, 2020-07
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    98
    New England Biolabs t4 rna ligase 2 truncated kq
    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using <t>T4</t> RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    T4 Rna Ligase 2 Truncated Kq, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 2 truncated kq/product/New England Biolabs
    Average 98 stars, based on 121 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 truncated kq - by Bioz Stars, 2020-07
    98/100 stars
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    Image Search Results


    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

    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

    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: In the experiments where different ligases were investigated, T4 RNA Ligase 2 truncated, T4 RNA Ligase 2 truncated R55K K227Q, and Thermostable 5′ App DNA/RNA Ligase were all obtained from New England Biolabs.

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    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

    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: Ligation Protocol Unless otherwise indicated, ligation was performed by mixing 1.25 µL of 2 µM adenylated adapter, 1 µL of T4 RNA Ligase buffer (New England Biolabs, Ipswich, MA), 5 µL of 50% PEG8000, 1 µL of synthetic target, 0.5 µL of total RNA, 1 µL of T4 RNA Ligase 2 truncated K227Q (New England Biolabs, Ipswich, MA) and water into a 20 µL reaction volume.

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.

    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: Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.

    Article Snippet: Ligation Protocol Unless otherwise indicated, ligation was performed by mixing 1.25 µL of 2 µM adenylated adapter, 1 µL of T4 RNA Ligase buffer (New England Biolabs, Ipswich, MA), 5 µL of 50% PEG8000, 1 µL of synthetic target, 0.5 µL of total RNA, 1 µL of T4 RNA Ligase 2 truncated K227Q (New England Biolabs, Ipswich, MA) and water into a 20 µL reaction volume.

    Techniques: Ligation, Labeling

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Journal: PLoS ONE

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    doi: 10.1371/journal.pone.0167009

    Figure Lengend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Article Snippet: Ligation Step 1: 1X Buffer 1 (50 mM Tris(hydroxymethyl)aminomethane HCl pH 7.5, 10 mM MgCl2 , 1 mM dithiothreital, ~20% polyethylene glycol (PEG) 8000) (TriLink Biotechnologies), 0.5 μM CleanTag 3΄ Adapter (TriLink Biotechnologies, LLC.), 40 Units murine RNase Inhibitor (New England Biolabs), 200 Units of T4 RNA Ligase 2 truncated KQ (New England Biolabs), RNA input (1 μg), 10 μL total volume.

    Techniques: Ligation, Modification