t4 rna ligase 2 truncated kq  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    T4 RNA Ligase 2 truncated KQ
    Description:
    T4 RNA Ligase 2 truncated KQ 10 000 units
    Catalog Number:
    m0373l
    Price:
    278
    Size:
    10 000 units
    Category:
    RNA Ligases
    Buy from Supplier


    Structured Review

    New England Biolabs t4 rna ligase 2 truncated kq
    T4 RNA Ligase 2 truncated KQ
    T4 RNA Ligase 2 truncated KQ 10 000 units
    https://www.bioz.com/result/t4 rna ligase 2 truncated kq/product/New England Biolabs
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 truncated kq - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    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.
    Figure Legend 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.

    Techniques Used: Ligation, Modification

    2) Product Images from "Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq"

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

    Journal: bioRxiv

    doi: 10.1101/2020.08.10.243931

    TrAEL-seq accurately maps and quantifies 3’ ends of DNA A : Schematic representation of TrAEL-seq method. Agarose-embedded genomic DNA is used as a starting material, plugs are washed extensively to remove unligated TrAEL adaptor 1 and agarose is removed prior to Bst 2.0 polymerase step. The blunting and ligation of TrAEL-adaptor 2 is performed using a NEBNext Ultra II DNA kit, and TrAEL-adaptor 2 homodimers removed by washing streptavidin beads before USER enzyme treatment. The finished material is ready for PCR amplification and sequencing using the NEBNext amplification system. Note that TrAEL-seq reads map antisense to the cleaved strand, reading the complementary sequence starting from the first nucleotide before the cleavage site. * - biotin moiety, U - deoxyuracil, N - any DNA base, rA - adenosine. B : in vitro assay of adaptor ligation. An 18 bp single stranded DNA oligonucleotide was treated with or without TdT then ligated to TrAEL adaptor 1 using T4 RNA ligase 2 truncated KQ. Products were separated on a 15% PAGE gel and visualised by SYBR Gold staining. C : Scatter plot comparing read counts from Sfi I digested DNA and the genome average based on END-seq and TrAEL-seq, showing similar signal-to-noise ratio for both methods. Note that the genome average signal encompasses all single-copy 1kb regions that do not overlap with an Sfi I site, while Sfi I quantitation represents reads mapping to the 13bp recognition site. Direct comparison of read counts between the Sfi I and the genome average probes is misleading because of the difference in probe size. An accurate comparison of this type is not possible as the Sfi I cleavages are spread over 3bp (see Fig. 1D ), and the majority of uncleaved 3bp sites in the genome have no mapped reads. In reality, the signal from the cleaved sites is at least 10 log2 units greater than an equivalent uncleaved region of the genome measured by either END-seq or TrAEL-seq. D : Precision mapping of Sfi I cleavage sites by TrAEL-seq and END-seq. Sfi I sites, which contain 5 degenerate bases were split into those that contain no A’s at the cleavage site (GGCCNNNB|BGGCC, 87 sites, upper panel) or A’s flanking the cleavage site (GGCCNNNA|AGGCC, 15 sites, lower panel), considering cleavage sites on forward and reverse strands separately. Mapped locations of 3’ ends were averaged across each category of site and expressed as a percentage of all 3’ ends mapped by each method to that category of site. E : Comparison of meiotic DSB profiles from dmc1 Δ cells performed by TrAEL-seq and sea2 Δ cells by S1-seq (SRA accession: SRP261135). Both techniques should map Spo11 cleavage sites in the given mutants. 25kb and 2.5kb regions of chromosome III are shown for reads counted in 20bp windows. F: Scatter plots of log transformed normalised read counts at all 3907 Spo11 cleavage hotspots annotated by Mohibullah and Keeney, comparing dmc1 Δ TrAEL-seq with either sae2 Δ S1-seq (left) or sequencing data for Spo11-associated oligonucleotides (right) [ 16 , 42 , 43 , 83 ], (SRA accessions: SRP261135 and SRR1976210 respectively)
    Figure Legend Snippet: TrAEL-seq accurately maps and quantifies 3’ ends of DNA A : Schematic representation of TrAEL-seq method. Agarose-embedded genomic DNA is used as a starting material, plugs are washed extensively to remove unligated TrAEL adaptor 1 and agarose is removed prior to Bst 2.0 polymerase step. The blunting and ligation of TrAEL-adaptor 2 is performed using a NEBNext Ultra II DNA kit, and TrAEL-adaptor 2 homodimers removed by washing streptavidin beads before USER enzyme treatment. The finished material is ready for PCR amplification and sequencing using the NEBNext amplification system. Note that TrAEL-seq reads map antisense to the cleaved strand, reading the complementary sequence starting from the first nucleotide before the cleavage site. * - biotin moiety, U - deoxyuracil, N - any DNA base, rA - adenosine. B : in vitro assay of adaptor ligation. An 18 bp single stranded DNA oligonucleotide was treated with or without TdT then ligated to TrAEL adaptor 1 using T4 RNA ligase 2 truncated KQ. Products were separated on a 15% PAGE gel and visualised by SYBR Gold staining. C : Scatter plot comparing read counts from Sfi I digested DNA and the genome average based on END-seq and TrAEL-seq, showing similar signal-to-noise ratio for both methods. Note that the genome average signal encompasses all single-copy 1kb regions that do not overlap with an Sfi I site, while Sfi I quantitation represents reads mapping to the 13bp recognition site. Direct comparison of read counts between the Sfi I and the genome average probes is misleading because of the difference in probe size. An accurate comparison of this type is not possible as the Sfi I cleavages are spread over 3bp (see Fig. 1D ), and the majority of uncleaved 3bp sites in the genome have no mapped reads. In reality, the signal from the cleaved sites is at least 10 log2 units greater than an equivalent uncleaved region of the genome measured by either END-seq or TrAEL-seq. D : Precision mapping of Sfi I cleavage sites by TrAEL-seq and END-seq. Sfi I sites, which contain 5 degenerate bases were split into those that contain no A’s at the cleavage site (GGCCNNNB|BGGCC, 87 sites, upper panel) or A’s flanking the cleavage site (GGCCNNNA|AGGCC, 15 sites, lower panel), considering cleavage sites on forward and reverse strands separately. Mapped locations of 3’ ends were averaged across each category of site and expressed as a percentage of all 3’ ends mapped by each method to that category of site. E : Comparison of meiotic DSB profiles from dmc1 Δ cells performed by TrAEL-seq and sea2 Δ cells by S1-seq (SRA accession: SRP261135). Both techniques should map Spo11 cleavage sites in the given mutants. 25kb and 2.5kb regions of chromosome III are shown for reads counted in 20bp windows. F: Scatter plots of log transformed normalised read counts at all 3907 Spo11 cleavage hotspots annotated by Mohibullah and Keeney, comparing dmc1 Δ TrAEL-seq with either sae2 Δ S1-seq (left) or sequencing data for Spo11-associated oligonucleotides (right) [ 16 , 42 , 43 , 83 ], (SRA accessions: SRP261135 and SRR1976210 respectively)

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Sequencing, In Vitro, Polyacrylamide Gel Electrophoresis, Staining, Quantitation Assay, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs
    Article Snippet: .. After RNA isolation of FLAG-mutant Dis3l2 IP samples, 100 ng RNAs were ligated overnight at 25 °C to 2 μM Universal miRNA Cloning Linker (NEB) using 200 units of T4 RNA ligase 2 truncated KQ (NEB) at the presence of 25% PEG 8000, and RNaseOUT. .. Ligated RNAs were purified using RNA Clean & Concentrator-25 columns (Zymo Research) and reverse transcribed by SuperScript III (see RNA extraction and qRT-PCR section) and universal RT+linker primer ( ). cDNAs were diluted and 100 ng cDNA was used for PCR reaction using U1 and U2 gene-specific forward primers ( ) and universal RT+linker reverse primer.

    High Throughput Screening Assay:

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: .. PAR-CLIP library preparation and high-throughput sequencing For 3′ adaptor ligation, beads were resuspended in 1 × T4 RNA ligase buffer (NEB) containing 10 U/µL T4 RNA ligase 2 (KQ) (NEB, M0373), 10 μM 3′ adaptor (5′ 5rApp-TGGAATTCTCGGGTGCCAAGG-3ddC 3′ (IDT, Inc., Coralville, IA )) , 1 U/µL RNase OUT, and 15% (w/v) PEG 8000. ..

    Ligation:

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: .. PAR-CLIP library preparation and high-throughput sequencing For 3′ adaptor ligation, beads were resuspended in 1 × T4 RNA ligase buffer (NEB) containing 10 U/µL T4 RNA ligase 2 (KQ) (NEB, M0373), 10 μM 3′ adaptor (5′ 5rApp-TGGAATTCTCGGGTGCCAAGG-3ddC 3′ (IDT, Inc., Coralville, IA )) , 1 U/µL RNase OUT, and 15% (w/v) PEG 8000. ..

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

    Isolation:

    Article Title: Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs
    Article Snippet: .. After RNA isolation of FLAG-mutant Dis3l2 IP samples, 100 ng RNAs were ligated overnight at 25 °C to 2 μM Universal miRNA Cloning Linker (NEB) using 200 units of T4 RNA ligase 2 truncated KQ (NEB) at the presence of 25% PEG 8000, and RNaseOUT. .. Ligated RNAs were purified using RNA Clean & Concentrator-25 columns (Zymo Research) and reverse transcribed by SuperScript III (see RNA extraction and qRT-PCR section) and universal RT+linker primer ( ). cDNAs were diluted and 100 ng cDNA was used for PCR reaction using U1 and U2 gene-specific forward primers ( ) and universal RT+linker reverse primer.

    Sequencing:

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: .. PAR-CLIP library preparation and high-throughput sequencing For 3′ adaptor ligation, beads were resuspended in 1 × T4 RNA ligase buffer (NEB) containing 10 U/µL T4 RNA ligase 2 (KQ) (NEB, M0373), 10 μM 3′ adaptor (5′ 5rApp-TGGAATTCTCGGGTGCCAAGG-3ddC 3′ (IDT, Inc., Coralville, IA )) , 1 U/µL RNase OUT, and 15% (w/v) PEG 8000. ..

    Incubation:

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq
    Article Snippet: .. Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000, 1 μl 10 pM/μl TrAEL-seq adaptor 1 and 1 μl T4 RNA ligase 2 truncated KQ (NEB M0373L). ..

    Article Title: A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies
    Article Snippet: .. The denatured sample was then incubated with 1 μL T4 RNA Ligase 2, truncated KQ (NEB, M0373S), 1 μL 10x buffer, 6 μL 50% PEG 8000, and 1.5 μL water for 4.5 h at 25°C. .. Free adaptor was then removed by addition of 1 μL 10 U/μL 5’-Deadenylase (NEB, M0331S).

    other:

    Article Title: mRNA and Small RNA-seq Reveal Insights into Immune Regulation in Apis cerana after Chinese Sacbrood Virus Infection
    Article Snippet: Since the small RNA has a phosphate and hydroxyl group at the 5’ and 3’ end, respectively, the T4 RNA ligase 1 and ligase 2 (truncated) were respectively ligated to corresponding ends of the small RNA.

    IA:

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: .. PAR-CLIP library preparation and high-throughput sequencing For 3′ adaptor ligation, beads were resuspended in 1 × T4 RNA ligase buffer (NEB) containing 10 U/µL T4 RNA ligase 2 (KQ) (NEB, M0373), 10 μM 3′ adaptor (5′ 5rApp-TGGAATTCTCGGGTGCCAAGG-3ddC 3′ (IDT, Inc., Coralville, IA )) , 1 U/µL RNase OUT, and 15% (w/v) PEG 8000. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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: 99/100, based on 18 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 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 truncated kq - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 rna ligase 2
    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, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 205 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 205 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

    TrAEL-seq accurately maps and quantifies 3’ ends of DNA A : Schematic representation of TrAEL-seq method. Agarose-embedded genomic DNA is used as a starting material, plugs are washed extensively to remove unligated TrAEL adaptor 1 and agarose is removed prior to Bst 2.0 polymerase step. The blunting and ligation of TrAEL-adaptor 2 is performed using a NEBNext Ultra II DNA kit, and TrAEL-adaptor 2 homodimers removed by washing streptavidin beads before USER enzyme treatment. The finished material is ready for PCR amplification and sequencing using the NEBNext amplification system. Note that TrAEL-seq reads map antisense to the cleaved strand, reading the complementary sequence starting from the first nucleotide before the cleavage site. * - biotin moiety, U - deoxyuracil, N - any DNA base, rA - adenosine. B : in vitro assay of adaptor ligation. An 18 bp single stranded DNA oligonucleotide was treated with or without TdT then ligated to TrAEL adaptor 1 using T4 RNA ligase 2 truncated KQ. Products were separated on a 15% PAGE gel and visualised by SYBR Gold staining. C : Scatter plot comparing read counts from Sfi I digested DNA and the genome average based on END-seq and TrAEL-seq, showing similar signal-to-noise ratio for both methods. Note that the genome average signal encompasses all single-copy 1kb regions that do not overlap with an Sfi I site, while Sfi I quantitation represents reads mapping to the 13bp recognition site. Direct comparison of read counts between the Sfi I and the genome average probes is misleading because of the difference in probe size. An accurate comparison of this type is not possible as the Sfi I cleavages are spread over 3bp (see Fig. 1D ), and the majority of uncleaved 3bp sites in the genome have no mapped reads. In reality, the signal from the cleaved sites is at least 10 log2 units greater than an equivalent uncleaved region of the genome measured by either END-seq or TrAEL-seq. D : Precision mapping of Sfi I cleavage sites by TrAEL-seq and END-seq. Sfi I sites, which contain 5 degenerate bases were split into those that contain no A’s at the cleavage site (GGCCNNNB|BGGCC, 87 sites, upper panel) or A’s flanking the cleavage site (GGCCNNNA|AGGCC, 15 sites, lower panel), considering cleavage sites on forward and reverse strands separately. Mapped locations of 3’ ends were averaged across each category of site and expressed as a percentage of all 3’ ends mapped by each method to that category of site. E : Comparison of meiotic DSB profiles from dmc1 Δ cells performed by TrAEL-seq and sea2 Δ cells by S1-seq (SRA accession: SRP261135). Both techniques should map Spo11 cleavage sites in the given mutants. 25kb and 2.5kb regions of chromosome III are shown for reads counted in 20bp windows. F: Scatter plots of log transformed normalised read counts at all 3907 Spo11 cleavage hotspots annotated by Mohibullah and Keeney, comparing dmc1 Δ TrAEL-seq with either sae2 Δ S1-seq (left) or sequencing data for Spo11-associated oligonucleotides (right) [ 16 , 42 , 43 , 83 ], (SRA accessions: SRP261135 and SRR1976210 respectively)

    Journal: bioRxiv

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

    doi: 10.1101/2020.08.10.243931

    Figure Lengend Snippet: TrAEL-seq accurately maps and quantifies 3’ ends of DNA A : Schematic representation of TrAEL-seq method. Agarose-embedded genomic DNA is used as a starting material, plugs are washed extensively to remove unligated TrAEL adaptor 1 and agarose is removed prior to Bst 2.0 polymerase step. The blunting and ligation of TrAEL-adaptor 2 is performed using a NEBNext Ultra II DNA kit, and TrAEL-adaptor 2 homodimers removed by washing streptavidin beads before USER enzyme treatment. The finished material is ready for PCR amplification and sequencing using the NEBNext amplification system. Note that TrAEL-seq reads map antisense to the cleaved strand, reading the complementary sequence starting from the first nucleotide before the cleavage site. * - biotin moiety, U - deoxyuracil, N - any DNA base, rA - adenosine. B : in vitro assay of adaptor ligation. An 18 bp single stranded DNA oligonucleotide was treated with or without TdT then ligated to TrAEL adaptor 1 using T4 RNA ligase 2 truncated KQ. Products were separated on a 15% PAGE gel and visualised by SYBR Gold staining. C : Scatter plot comparing read counts from Sfi I digested DNA and the genome average based on END-seq and TrAEL-seq, showing similar signal-to-noise ratio for both methods. Note that the genome average signal encompasses all single-copy 1kb regions that do not overlap with an Sfi I site, while Sfi I quantitation represents reads mapping to the 13bp recognition site. Direct comparison of read counts between the Sfi I and the genome average probes is misleading because of the difference in probe size. An accurate comparison of this type is not possible as the Sfi I cleavages are spread over 3bp (see Fig. 1D ), and the majority of uncleaved 3bp sites in the genome have no mapped reads. In reality, the signal from the cleaved sites is at least 10 log2 units greater than an equivalent uncleaved region of the genome measured by either END-seq or TrAEL-seq. D : Precision mapping of Sfi I cleavage sites by TrAEL-seq and END-seq. Sfi I sites, which contain 5 degenerate bases were split into those that contain no A’s at the cleavage site (GGCCNNNB|BGGCC, 87 sites, upper panel) or A’s flanking the cleavage site (GGCCNNNA|AGGCC, 15 sites, lower panel), considering cleavage sites on forward and reverse strands separately. Mapped locations of 3’ ends were averaged across each category of site and expressed as a percentage of all 3’ ends mapped by each method to that category of site. E : Comparison of meiotic DSB profiles from dmc1 Δ cells performed by TrAEL-seq and sea2 Δ cells by S1-seq (SRA accession: SRP261135). Both techniques should map Spo11 cleavage sites in the given mutants. 25kb and 2.5kb regions of chromosome III are shown for reads counted in 20bp windows. F: Scatter plots of log transformed normalised read counts at all 3907 Spo11 cleavage hotspots annotated by Mohibullah and Keeney, comparing dmc1 Δ TrAEL-seq with either sae2 Δ S1-seq (left) or sequencing data for Spo11-associated oligonucleotides (right) [ 16 , 42 , 43 , 83 ], (SRA accessions: SRP261135 and SRR1976210 respectively)

    Article Snippet: Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000, 1 μl 10 pM/μl TrAEL-seq adaptor 1 and 1 μl T4 RNA ligase 2 truncated KQ (NEB M0373L).

    Techniques: Ligation, Polymerase Chain Reaction, Amplification, Sequencing, In Vitro, Polyacrylamide Gel Electrophoresis, Staining, Quantitation Assay, Transformation Assay

    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