ligation  (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 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    m0202l
    Price:
    256
    Size:
    100 000 units
    Category:
    DNA Ligases
    Buy from Supplier


    Structured Review

    New England Biolabs ligation
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/ligation/product/New England Biolabs
    Average 99 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    ligation - by Bioz Stars, 2020-09
    99/100 stars

    Images

    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
    Article Snippet: .. The reaction was supplemented with T4 DNA ligase (80 U) in buffer (50 mM Tris·HCl pH 7.6, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) and incubated at 16 °C for 12 h. The mixture was supplemented with 6X loading buffer (NEB), heated at 75 °C for 15 min and separated using preparative 0.8% w/v agarose gel electrophoresis containing 0.5 µg/mL EtdBr. ..

    Ligation:

    Article Title: Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils
    Article Snippet: .. A ligation solution (20 μl) containing 20 mM Tris-HCl (pH 7.4)–50 mM KCl–10 mM MgCl2 –1 mM DTT–3 mM ATP–200 U of T4 DNA ligase (New England Biolabs)–100 μg of BSA per ml was added to each mixture. .. The ligation reaction was for 15 min at 22°C and was stopped by the addition of SDS and EDTA to 0.6% and 30 mM, respectively.

    Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer
    Article Snippet: .. H2 O2 -stressed cells were treated with formaldehyde to obtain cross-linked chromatin, after which DNA/protein complexes were first digested with the Csp6I restriction enzyme and then selected with anti-HSF1 antibodies; at last, ligation of chromatin fragments was carried out with the T4 DNA Ligase (Figure ). .. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of ligated chromatin fragments (Figure ).

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    Mutagenesis:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    Produced:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    Incubation:

    Article Title: Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver
    Article Snippet: .. After digestion, the DNA mixture was incubated at 65°C for 20 min, and the DNA was self-ligated with T4 DNA ligase (New England Biolabs) at 16°C overnight in 700 μl of reaction mixture. .. The DNA was extracted with phenol-chloroform, precipitated by isopropanol , and resuspended, and 2.25 μg was used to transform the bacteria by electroporation.

    Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
    Article Snippet: .. The reaction was supplemented with T4 DNA ligase (80 U) in buffer (50 mM Tris·HCl pH 7.6, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) and incubated at 16 °C for 12 h. The mixture was supplemented with 6X loading buffer (NEB), heated at 75 °C for 15 min and separated using preparative 0.8% w/v agarose gel electrophoresis containing 0.5 µg/mL EtdBr. ..

    other:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: For this, we performed the POLRMT-primed minicircle assay with T4 DNA ligase, and then tested whether the closed circular products could be subsequently converted to nicked circles by KOH treatment ( ).

    Article Title: Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity
    Article Snippet: In contrast, the electrophoretic mobility of mtDNA from control cells was unaffected by treatment with T4 DNA ligase.

    Article Title: Ligation with Nucleic Acid Sequence-Based Amplification
    Article Snippet: Only when P3, P5, Target, T4 DNA Ligase, and the two primers were present was a 72-nt band observed.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    New England Biolabs ligation reactions in vitro ligation reactions
    Scheme of <t>in</t> <t>vitro</t> <t>ligation</t> selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.
    Ligation Reactions In Vitro Ligation Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ligation reactions in vitro ligation reactions/product/New England Biolabs
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ligation reactions in vitro ligation reactions - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    88
    New England Biolabs end ligation
    Related to Figure 5. α-globin genes exhibit readthrough transcription (A) Individual long reads are shown for the α-globin 1 gene ( Hba-a1 ) and α-globin 2 gene ( Hba-a2 ). Diagrams are as described in Figure 2 . Data represent two biological replicates and two technical replicates combined. (B-C) PRO-seq <t>3′</t> end read coverage is shown downstream of the Hba-a1 (B) and Hba-a2 (C) gene loci. We note that the duplicated copies of α-globin in the genome ( Hba-a1 and Hba-a2 ) impedes unique mapping of short PRO-seq reads in the coding sequence, artificially reducing gene body reads. Red dotted line indicates PAS. Data represent three biological replicates combined.
    End Ligation, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/end ligation/product/New England Biolabs
    Average 88 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    end ligation - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    85
    New England Biolabs restriction ligation cloning restriction endonucleases bsteii
    Integration of <t>polyprotein</t> expression cassettes into MultiBac baculovirus. ( a ) MultiBac Acceptor vectors pPBac, pKL-PBac, and pOmni-PBac, tailored for polyprotein production, are shown schematically ( top ). They contain a regular ColE1 origin of replication and a polyprotein expression cassette, which encodes an N-terminal TEV protease and a C-terminal CFP spaced by a TEV cleavage site (tcs). <t>BstEII</t> and RsrII sites are used for inserting the polyprotein encoding ORF of interest. Donor vectors pIDC, pIDK, and pIDS contain a conditional origin of replication derived from the R6Kγ phage [ 13 ]. The multiplication module flanking the expression cassettes contain a homing endonuclease site and a complementary BstXI site ( boxes in light blue ). Polh and p10 are baculoviral very late promoters; SV40 and HSVtk are polyadenylation signals. MCS1 and MCS2 stand for multiple cloning sites. Tn7L and Tn7R are specific DNA sequences for Tn7 transposition; the lef2/603 and Ori1629 homology regions are shown as gray boxes . LoxP sites are shown as red balls . Cm stands for chloramphenicol, Gn for gentamicin, Kn for kanamycin, Sp for spectinomycin. ( b ) Besides polyprotein expression cassettes, single protein and multigene expression cassettes can also be integrated into the Tn7 attachment site (mini-attTn7) harbored by the LacZ (lacZα) gene, or the LoxP site of the MultiBac baculovirus. Ap stands for ampicillin. The F-Replicon is a single-copy bacterial origin of replication. For reagents contact: iberger@embl.fr
    Restriction Ligation Cloning Restriction Endonucleases Bsteii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction ligation cloning restriction endonucleases bsteii/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction ligation cloning restriction endonucleases bsteii - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: In Vitro, Ligation, Selection, Sequencing, Amplification, Introduce

    Comparison of miRNA ligation efficiencies using SR1 versus SR1-S adapter. ( A ) Sequences and predicted secondary structures of SR1 and SR1-S adapters. The underlined sequence is shared by both adapters. The secondary structures of SR1 and SR1-S are presented in bracket and dot form where brackets represent base paired nucleotides and dots represent unpaired nucleotides. ( B ) Ligation reactions of miRNAs with the SR1 or SR1-S adapter were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold. ( C ) Ligation efficiency was calculated and plotted. The data points plotted represent average ligation efficiency ± standard deviation from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Comparison of miRNA ligation efficiencies using SR1 versus SR1-S adapter. ( A ) Sequences and predicted secondary structures of SR1 and SR1-S adapters. The underlined sequence is shared by both adapters. The secondary structures of SR1 and SR1-S are presented in bracket and dot form where brackets represent base paired nucleotides and dots represent unpaired nucleotides. ( B ) Ligation reactions of miRNAs with the SR1 or SR1-S adapter were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold. ( C ) Ligation efficiency was calculated and plotted. The data points plotted represent average ligation efficiency ± standard deviation from two independent experiments.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Sequencing, Staining, Standard Deviation

    Improving miRNA ligation efficiency using redesigned adapters. ( A ) Ligation of miRNAs with SR1 adapter or a new adapter specifically designed for each miRNA. Ligation reactions were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels, stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiency was determined and plotted. The data points represent average ligation efficiency ± standard deviation from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improving miRNA ligation efficiency using redesigned adapters. ( A ) Ligation of miRNAs with SR1 adapter or a new adapter specifically designed for each miRNA. Ligation reactions were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels, stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiency was determined and plotted. The data points represent average ligation efficiency ± standard deviation from two independent experiments.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Staining, Standard Deviation

    Improvement of miRNA ligation efficiency using a randomized adapter in the presence of mouse ES cell small RNAs. ( A ) Scheme of ligation reactions in the presence of mouse ES cell small RNAs. Each reaction contained 0.75 fmol of a 5′- 32 P labeled miRNA mixed with a 500-fold excess of mouse ES cell small RNAs and either the SR1 or the SR1-R adapter. Gray lines represent the ES cell small RNAs and the black line with an asterisk represents the radio-labeled miRNA. The SR1-R adapter is shown in black with a wavy line representing the random region at the 5′-end. Ligation products were resolved on 15% TBE–urea acrylamide gels, exposed to phosphor storage screens, and scanned. The ligated radio-labeled miRNA appears as a higher molecular weight band than unligated miRNA. ( B ) Representative results of ligation gels as described in A. ( C ) Comparison of ligation efficiency of miRNAs with the SR1 and SR1-R adapters. The intensity of ligated and unligated bands in each lane was quantified and ligation efficiencies were determined by calculating the percentage of ligated miRNA from the total miRNA. The data are represented as the average ± standard deviation ligation efficiency from two independent experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improvement of miRNA ligation efficiency using a randomized adapter in the presence of mouse ES cell small RNAs. ( A ) Scheme of ligation reactions in the presence of mouse ES cell small RNAs. Each reaction contained 0.75 fmol of a 5′- 32 P labeled miRNA mixed with a 500-fold excess of mouse ES cell small RNAs and either the SR1 or the SR1-R adapter. Gray lines represent the ES cell small RNAs and the black line with an asterisk represents the radio-labeled miRNA. The SR1-R adapter is shown in black with a wavy line representing the random region at the 5′-end. Ligation products were resolved on 15% TBE–urea acrylamide gels, exposed to phosphor storage screens, and scanned. The ligated radio-labeled miRNA appears as a higher molecular weight band than unligated miRNA. ( B ) Representative results of ligation gels as described in A. ( C ) Comparison of ligation efficiency of miRNAs with the SR1 and SR1-R adapters. The intensity of ligated and unligated bands in each lane was quantified and ligation efficiencies were determined by calculating the percentage of ligated miRNA from the total miRNA. The data are represented as the average ± standard deviation ligation efficiency from two independent experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Labeling, Molecular Weight, Standard Deviation

    Improvement of miRNA ligation efficiencies using a randomized adapter, SR1-R. ( A ) Ligation reactions were performed with Rnl2tr and the SR1 or SR1-R adapter. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiencies of 24 miRNAs with the SR1 or SR1-R adapter were determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improvement of miRNA ligation efficiencies using a randomized adapter, SR1-R. ( A ) Ligation reactions were performed with Rnl2tr and the SR1 or SR1-R adapter. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiencies of 24 miRNAs with the SR1 or SR1-R adapter were determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Staining, Standard Deviation

    3′-adapter ligation efficiencies of miRNAs. ( A ) Each miRNA was incubated in a ligation reaction containing Rnl2tr with or without SR1 adapter. The ligation products were separated on 15% TBE–urea gels and visualized with SYBR Gold. Ligated products correspond to high molecular weight bands, which only appear in reactions with SR1 adapter. Unligated miRNAs and SR1 adapters remain as lower molecular weight bands. ( B ) The ligation efficiency of each miRNA was determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: 3′-adapter ligation efficiencies of miRNAs. ( A ) Each miRNA was incubated in a ligation reaction containing Rnl2tr with or without SR1 adapter. The ligation products were separated on 15% TBE–urea gels and visualized with SYBR Gold. Ligated products correspond to high molecular weight bands, which only appear in reactions with SR1 adapter. Unligated miRNAs and SR1 adapters remain as lower molecular weight bands. ( B ) The ligation efficiency of each miRNA was determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Incubation, Molecular Weight, Standard Deviation

    Related to Figure 5. α-globin genes exhibit readthrough transcription (A) Individual long reads are shown for the α-globin 1 gene ( Hba-a1 ) and α-globin 2 gene ( Hba-a2 ). Diagrams are as described in Figure 2 . Data represent two biological replicates and two technical replicates combined. (B-C) PRO-seq 3′ end read coverage is shown downstream of the Hba-a1 (B) and Hba-a2 (C) gene loci. We note that the duplicated copies of α-globin in the genome ( Hba-a1 and Hba-a2 ) impedes unique mapping of short PRO-seq reads in the coding sequence, artificially reducing gene body reads. Red dotted line indicates PAS. Data represent three biological replicates combined.

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Related to Figure 5. α-globin genes exhibit readthrough transcription (A) Individual long reads are shown for the α-globin 1 gene ( Hba-a1 ) and α-globin 2 gene ( Hba-a2 ). Diagrams are as described in Figure 2 . Data represent two biological replicates and two technical replicates combined. (B-C) PRO-seq 3′ end read coverage is shown downstream of the Hba-a1 (B) and Hba-a2 (C) gene loci. We note that the duplicated copies of α-globin in the genome ( Hba-a1 and Hba-a2 ) impedes unique mapping of short PRO-seq reads in the coding sequence, artificially reducing gene body reads. Red dotted line indicates PAS. Data represent three biological replicates combined.

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques: Sequencing

    Long read sequencing reveals a range of co-transcriptional splicing efficiencies (A) Illustration of LRS data. Gene diagram is shown at the top, with the black arrow indicating the TSS. Reads are aligned to the genome and ordered by 3′ end position. Each read is colored by its splicing status: reads that cross one or more introns but contain no splice junction are “all unspliced” (yellow); reads that cross one or more intron and are spliced at every intron are “all spliced” (dark purple); reads that cross two or more introns but are not spliced at all introns are “partially spliced” (light purple); and reads that do not span an entire intron are designated “NA” (gray). Each horizontal row represents one read. Regions of missing sequence (e.g. spliced introns) are transparent. Light gray shading indicates regions of exons, and dark gray shading indicates the region downstream of the annotated PAS (dotted red line). N is the number of individual long reads aligned to each gene, and coSE is the calculated co-transcriptional splicing efficiency (coSE) for each gene; coSE is defined as the number of spliced introns/(spliced introns + unspliced introns) across all reads aligned to a gene. (B,C) LRS data are shown for uninduced (top) and induced (bottom) MEL cells at four representative genes: (B) protein-coding genes Pabpc1 , Actb , and Calr , and (C) the lncRNA Snhg5 . (D) Fraction of introns spliced co-transcriptionally. Only introns from genes with ≥ 10 aligned reads and introns less than 5 kb in uninduced and induced conditions are considered (n = 1,210 genes; 15,911 introns) (E) Per-gene coSE in uninduced and induced conditions. Each point represents a single gene as defined in (D) . R 2 is Pearson correlation coefficient. For (B) - (E) , data represent two biological replicates and two technical replicates combined. See also Figure S3 .

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Long read sequencing reveals a range of co-transcriptional splicing efficiencies (A) Illustration of LRS data. Gene diagram is shown at the top, with the black arrow indicating the TSS. Reads are aligned to the genome and ordered by 3′ end position. Each read is colored by its splicing status: reads that cross one or more introns but contain no splice junction are “all unspliced” (yellow); reads that cross one or more intron and are spliced at every intron are “all spliced” (dark purple); reads that cross two or more introns but are not spliced at all introns are “partially spliced” (light purple); and reads that do not span an entire intron are designated “NA” (gray). Each horizontal row represents one read. Regions of missing sequence (e.g. spliced introns) are transparent. Light gray shading indicates regions of exons, and dark gray shading indicates the region downstream of the annotated PAS (dotted red line). N is the number of individual long reads aligned to each gene, and coSE is the calculated co-transcriptional splicing efficiency (coSE) for each gene; coSE is defined as the number of spliced introns/(spliced introns + unspliced introns) across all reads aligned to a gene. (B,C) LRS data are shown for uninduced (top) and induced (bottom) MEL cells at four representative genes: (B) protein-coding genes Pabpc1 , Actb , and Calr , and (C) the lncRNA Snhg5 . (D) Fraction of introns spliced co-transcriptionally. Only introns from genes with ≥ 10 aligned reads and introns less than 5 kb in uninduced and induced conditions are considered (n = 1,210 genes; 15,911 introns) (E) Per-gene coSE in uninduced and induced conditions. Each point represents a single gene as defined in (D) . R 2 is Pearson correlation coefficient. For (B) - (E) , data represent two biological replicates and two technical replicates combined. See also Figure S3 .

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques: Sequencing

    Splicing can occur when elongating Pol II is just downstream of a newly transcribed 3 ′ splice site (A) Schematic defining the distance from the 3′ end of a nascent RNA (nRNA) to the most 3′-proximal splice junction. 3′ end sequence reports the position of Pol II when nascent RNA was isolated. (B) Distance (nt) from the 3′-most splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 300 nt past the 3′SS (n = 184,456 observations). (C) Distance (nt) from the 3′-most splice junction to Pol II position is shown categorized by gene biotype (n = 108,280 protein-coding genes, 440 pseudogenes, 90 antisense genes, 350 lincRNA genes). For (B) and (C) , data represent two biological replicates and two technical replicates in uninduced and induced cells combined. (D) Genome browser view showing spliced PRO-seq reads aligned to the Apbb1 gene, where 3′ ends of reads represent the position of elongating Pol II. Only spliced reads, filtered from all reads, are shown. (E) PRO-seq 3′ end coverage is shown aligned to active transcription start sites (TSS), 5′ splice sites (5′SS), and 3′ splice sites (3′SS). For (D) and (E) , data represent three biological replicates for uninduced and induced cells combined. See also Figure S4 .

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Splicing can occur when elongating Pol II is just downstream of a newly transcribed 3 ′ splice site (A) Schematic defining the distance from the 3′ end of a nascent RNA (nRNA) to the most 3′-proximal splice junction. 3′ end sequence reports the position of Pol II when nascent RNA was isolated. (B) Distance (nt) from the 3′-most splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 300 nt past the 3′SS (n = 184,456 observations). (C) Distance (nt) from the 3′-most splice junction to Pol II position is shown categorized by gene biotype (n = 108,280 protein-coding genes, 440 pseudogenes, 90 antisense genes, 350 lincRNA genes). For (B) and (C) , data represent two biological replicates and two technical replicates in uninduced and induced cells combined. (D) Genome browser view showing spliced PRO-seq reads aligned to the Apbb1 gene, where 3′ ends of reads represent the position of elongating Pol II. Only spliced reads, filtered from all reads, are shown. (E) PRO-seq 3′ end coverage is shown aligned to active transcription start sites (TSS), 5′ splice sites (5′SS), and 3′ splice sites (3′SS). For (D) and (E) , data represent three biological replicates for uninduced and induced cells combined. See also Figure S4 .

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques: Sequencing, Isolation

    Splicing intermediates are abundant at introns with weak 3 ′ splice sites (A) Schematic definition of first step splicing intermediates (dotted red oval), which have undergone the first step of splicing and have a free 3′-OH moiety that can be ligated to the 3′ end DNA adapter. Splicing intermediate reads are characterized by a 3′ end at the -1 position of a 5′SS (last nucleotide of the upstream exon). (B) Coverage of long read 3′ ends (top panels) and 5′ ends (bottom panels) aligned to all mm10 5′SSs (left) and 3′SSs (right). (C) Coverage of long read 3′ ends across four example genes: Alas2 , Hmbs , Hnrnpk , and Pkhd1l1 . Orange arrows indicate the positions where the most abundant splicing intermediates are observed in each gene. (D) Individual long reads are shown for the gene Alas2 . Diagram is similar to Figure 2 , except that individual reads are colored depending on whether they are splicing intermediates (orange) or not (gray). Data for uninduced and induced cells are shown combined. Potential recursive splicing site is indicated by an arrow; recursively spliced reads are shown in detail in (E) . (F) MaxEnt splice site scores for 5′SS (in purple) and 3′SS (in orange) for all introns is shown categorized by the normalized intermediate count (NIC) at each intron (n = the number of introns in each category). For (F) - (H) , NIC is defined as the number of splicing intermediate reads divided by the sum of the intermediate reads plus spliced reads at a 5′SS. (G) Sequence logos of the -20 to +3 nt region around the 3′SS used to calculate the 3′SS score in (F) . 3′SS dinucleotide is shown in dark gray, pyrimidines are shown in orange, and purines are shown in light gray. For (B) - (G) , data represent two biological replicates and two technical replicates from uninduced and induced cells combined. (H) PRO-seq 3′ end coverage aligned to 5′SSs, and 3′SSs for introns in three categories of NIC values (n = the number of introns in each category). Data represent three biological replicates for uninduced and induced cells combined. See also Figure S5 .

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Splicing intermediates are abundant at introns with weak 3 ′ splice sites (A) Schematic definition of first step splicing intermediates (dotted red oval), which have undergone the first step of splicing and have a free 3′-OH moiety that can be ligated to the 3′ end DNA adapter. Splicing intermediate reads are characterized by a 3′ end at the -1 position of a 5′SS (last nucleotide of the upstream exon). (B) Coverage of long read 3′ ends (top panels) and 5′ ends (bottom panels) aligned to all mm10 5′SSs (left) and 3′SSs (right). (C) Coverage of long read 3′ ends across four example genes: Alas2 , Hmbs , Hnrnpk , and Pkhd1l1 . Orange arrows indicate the positions where the most abundant splicing intermediates are observed in each gene. (D) Individual long reads are shown for the gene Alas2 . Diagram is similar to Figure 2 , except that individual reads are colored depending on whether they are splicing intermediates (orange) or not (gray). Data for uninduced and induced cells are shown combined. Potential recursive splicing site is indicated by an arrow; recursively spliced reads are shown in detail in (E) . (F) MaxEnt splice site scores for 5′SS (in purple) and 3′SS (in orange) for all introns is shown categorized by the normalized intermediate count (NIC) at each intron (n = the number of introns in each category). For (F) - (H) , NIC is defined as the number of splicing intermediate reads divided by the sum of the intermediate reads plus spliced reads at a 5′SS. (G) Sequence logos of the -20 to +3 nt region around the 3′SS used to calculate the 3′SS score in (F) . 3′SS dinucleotide is shown in dark gray, pyrimidines are shown in orange, and purines are shown in light gray. For (B) - (G) , data represent two biological replicates and two technical replicates from uninduced and induced cells combined. (H) PRO-seq 3′ end coverage aligned to 5′SSs, and 3′SSs for introns in three categories of NIC values (n = the number of introns in each category). Data represent three biological replicates for uninduced and induced cells combined. See also Figure S5 .

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques: Sequencing

    Poor splicing efficiency is associated with readthrough transcription (A) Individual long reads are shown for the major β-globin gene ( Hbb-b1 ). Diagram is as described in Figure 2 . (B) LRS coverage and PRO-seq 3′ end coverage in uninduced and induced cells is shown at the Hbb-b1 gene. Scale at the left indicates coverage in number of reads, and red dotted line indicates PAS. We note that the duplicated copies of β-globin in the genome ( Hbb-b1 and Hbb-b2 ) impedes unique mapping of short PRO-seq reads in the coding sequence, artificially reducing gene body reads. (C) Long read coverage in the region downstream of the last annotated PAS is shown normalized to coverage at the PAS. Unspliced coverage shows reads with no splice junctions (n = 626,130 reads), and spliced coverage shows reads with one or more splice junctions (n = 368,031 reads). Red dotted line indicates PAS. (D) Fraction of all reads (left) and readthrough reads (right) categorized by splicing status (as described in Figure 2 ). A readthrough read is categorized as having a 5′ end within a gene region and a 3′ end greater than 100 nt downstream of the gene end (n = number of reads in each category). For (A-D) , LRS data represent two biological replicates and two technical replicates combined, and PRO-seq data represent three biological replicates combined. For (D-E) , data from uninduced and induced cells are shown combined. See also Figure S6 .

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Poor splicing efficiency is associated with readthrough transcription (A) Individual long reads are shown for the major β-globin gene ( Hbb-b1 ). Diagram is as described in Figure 2 . (B) LRS coverage and PRO-seq 3′ end coverage in uninduced and induced cells is shown at the Hbb-b1 gene. Scale at the left indicates coverage in number of reads, and red dotted line indicates PAS. We note that the duplicated copies of β-globin in the genome ( Hbb-b1 and Hbb-b2 ) impedes unique mapping of short PRO-seq reads in the coding sequence, artificially reducing gene body reads. (C) Long read coverage in the region downstream of the last annotated PAS is shown normalized to coverage at the PAS. Unspliced coverage shows reads with no splice junctions (n = 626,130 reads), and spliced coverage shows reads with one or more splice junctions (n = 368,031 reads). Red dotted line indicates PAS. (D) Fraction of all reads (left) and readthrough reads (right) categorized by splicing status (as described in Figure 2 ). A readthrough read is categorized as having a 5′ end within a gene region and a 3′ end greater than 100 nt downstream of the gene end (n = number of reads in each category). For (A-D) , LRS data represent two biological replicates and two technical replicates combined, and PRO-seq data represent three biological replicates combined. For (D-E) , data from uninduced and induced cells are shown combined. See also Figure S6 .

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques: Sequencing

    Related to Figure 3. PRO-seq signal at 5 ′ SS is overlapping with TSS signal for short first introns (A) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 300 nt. Analysis is the same as in Figure 3B , but with reads from uninduced and induced cells plotted separately. (B) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown categorized by gene biotype. Analysis is the same as in Figure 3C , but with reads from uninduced and induced cells plotted separately. (C) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 1000 nt. Analysis is the same as in Figure 3B , but with data from this study and nanoCOP data from Drexler et al . (D) PRO-seq 3′ end coverage aligned to 5′SSs for all introns from active transcripts (dark purple), first introns only (light purple), and non-first introns (orange). Right panel shows data scaled to show all introns. (E) PRO-seq 3′ end coverage aligned to 5′SSs for all introns (dark purple), introns where the distance from the TSS to the 5′SS is ≤ 250 nt (light purple), and introns where the distance to the TSS to the 5′SS is > 250 nt (orange). Right panel shows data scaled to show all introns. (F) PRO-seq 3′ end coverage around splice sites separated by per-intron coSE. For each intron, coSE was calculated as the number of reads fully overlapping the intron that were spliced divided by the total number of reads fully overlapping the intron. For (A-B) , data represent two biological replicates and two technical replicates combined. For (C) , data from this study represent two biological replicates and two technical replicates from uninduced and induced cells combined. Data from Drexler et al. represent two samples in human BL1184 cells, 9 samples in Drosophila S2 cells, and 11 samples in human K562 cells. in or (D-F) data represent three biological replicates from uninduced and induced cells combined, and n = number of introns introns in each category.

    Journal: bioRxiv

    Article Title: Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

    doi: 10.1101/2020.02.11.944595

    Figure Lengend Snippet: Related to Figure 3. PRO-seq signal at 5 ′ SS is overlapping with TSS signal for short first introns (A) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 300 nt. Analysis is the same as in Figure 3B , but with reads from uninduced and induced cells plotted separately. (B) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown categorized by gene biotype. Analysis is the same as in Figure 3C , but with reads from uninduced and induced cells plotted separately. (C) Distance (nt) from the most 3′-proximal splice junction to Pol II position is shown as a cumulative fraction. Inset is a zoom in on the first 1000 nt. Analysis is the same as in Figure 3B , but with data from this study and nanoCOP data from Drexler et al . (D) PRO-seq 3′ end coverage aligned to 5′SSs for all introns from active transcripts (dark purple), first introns only (light purple), and non-first introns (orange). Right panel shows data scaled to show all introns. (E) PRO-seq 3′ end coverage aligned to 5′SSs for all introns (dark purple), introns where the distance from the TSS to the 5′SS is ≤ 250 nt (light purple), and introns where the distance to the TSS to the 5′SS is > 250 nt (orange). Right panel shows data scaled to show all introns. (F) PRO-seq 3′ end coverage around splice sites separated by per-intron coSE. For each intron, coSE was calculated as the number of reads fully overlapping the intron that were spliced divided by the total number of reads fully overlapping the intron. For (A-B) , data represent two biological replicates and two technical replicates combined. For (C) , data from this study represent two biological replicates and two technical replicates from uninduced and induced cells combined. Data from Drexler et al. represent two samples in human BL1184 cells, 9 samples in Drosophila S2 cells, and 11 samples in human K562 cells. in or (D-F) data represent three biological replicates from uninduced and induced cells combined, and n = number of introns introns in each category.

    Article Snippet: 3′ end ligation was performed using T4 RNA ligase I (NEB) for 2 hours at room temperature.

    Techniques:

    RADICL-seq method for the identification of RNA-chromatin interactions. a) Schematic representation of the RADICL-seq protocol. Top: sequence of enzymatic reactions taking place in fixed nuclei after partial lysis of the nuclear membrane. The adduct formed by genomic DNA (black), RNA (red) and proteins (blue circles) goes through controlled DNase digestion and chromatin preparation. After RNase H digestion, an adapter (dark blue) containing an internally biotinylated residue (black dot) bridges RNA and DNA in close proximity. Bottom: sequence of enzymatic reactions performed in solution. After reversal of crosslinks, the RNA-DNA chimera is converted into a fully dsDNA molecule and digested by EcoP15I enzyme to a designated length. After ligation of the sequencing linker and biotin pull-down, the library is PCR-amplified and high-throughput sequenced. b) Reproducibility of the RNA-DNA interaction frequencies across replicates, assessed by counting the occurrences of transcribed genes and 25 kb genomic bins pairs. c) RNA and d) DNA tags origin. The inner pie charts represent a broader classification into intergenic and genic (annotated genes), while the outer circles show a finer classification of the genic portion. e) Nuclear and f) cytosolic RNA-seq tags comparison with RADICL-seq RNA reads gene counts. The former are normalized to tags per million (TPM), while the latter are normalised to reads per kilobase (RPK). The linear regression lines are shown in red. g) Density of the normalized counts of DNA reads detected by RADICL-seq around ATAC-seq (red), DHS-seq (green) and H3K9me3 ChIP-seq (blue) peaks; dashed lines represent the density profiles of random genomic reads.

    Journal: bioRxiv

    Article Title: RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

    doi: 10.1101/681924

    Figure Lengend Snippet: RADICL-seq method for the identification of RNA-chromatin interactions. a) Schematic representation of the RADICL-seq protocol. Top: sequence of enzymatic reactions taking place in fixed nuclei after partial lysis of the nuclear membrane. The adduct formed by genomic DNA (black), RNA (red) and proteins (blue circles) goes through controlled DNase digestion and chromatin preparation. After RNase H digestion, an adapter (dark blue) containing an internally biotinylated residue (black dot) bridges RNA and DNA in close proximity. Bottom: sequence of enzymatic reactions performed in solution. After reversal of crosslinks, the RNA-DNA chimera is converted into a fully dsDNA molecule and digested by EcoP15I enzyme to a designated length. After ligation of the sequencing linker and biotin pull-down, the library is PCR-amplified and high-throughput sequenced. b) Reproducibility of the RNA-DNA interaction frequencies across replicates, assessed by counting the occurrences of transcribed genes and 25 kb genomic bins pairs. c) RNA and d) DNA tags origin. The inner pie charts represent a broader classification into intergenic and genic (annotated genes), while the outer circles show a finer classification of the genic portion. e) Nuclear and f) cytosolic RNA-seq tags comparison with RADICL-seq RNA reads gene counts. The former are normalized to tags per million (TPM), while the latter are normalised to reads per kilobase (RPK). The linear regression lines are shown in red. g) Density of the normalized counts of DNA reads detected by RADICL-seq around ATAC-seq (red), DHS-seq (green) and H3K9me3 ChIP-seq (blue) peaks; dashed lines represent the density profiles of random genomic reads.

    Article Snippet: Proximity ligation The in situ proximity ligation was carried out by resuspending the air-dried bead-nuclei mixture in 500 µl 1× T4 DNA Ligase Buffer with ATP containing 4 U/µl T4 DNA Ligase (New England Biolabs) and incubating it at room temperature for 4 h. After the incubation, bead-nuclei complexes were pelleted at 2,500g for 60 s and resuspended in 200 µl H2 O.

    Techniques: Sequencing, Lysis, Ligation, Polymerase Chain Reaction, Amplification, High Throughput Screening Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation

    Integration of polyprotein expression cassettes into MultiBac baculovirus. ( a ) MultiBac Acceptor vectors pPBac, pKL-PBac, and pOmni-PBac, tailored for polyprotein production, are shown schematically ( top ). They contain a regular ColE1 origin of replication and a polyprotein expression cassette, which encodes an N-terminal TEV protease and a C-terminal CFP spaced by a TEV cleavage site (tcs). BstEII and RsrII sites are used for inserting the polyprotein encoding ORF of interest. Donor vectors pIDC, pIDK, and pIDS contain a conditional origin of replication derived from the R6Kγ phage [ 13 ]. The multiplication module flanking the expression cassettes contain a homing endonuclease site and a complementary BstXI site ( boxes in light blue ). Polh and p10 are baculoviral very late promoters; SV40 and HSVtk are polyadenylation signals. MCS1 and MCS2 stand for multiple cloning sites. Tn7L and Tn7R are specific DNA sequences for Tn7 transposition; the lef2/603 and Ori1629 homology regions are shown as gray boxes . LoxP sites are shown as red balls . Cm stands for chloramphenicol, Gn for gentamicin, Kn for kanamycin, Sp for spectinomycin. ( b ) Besides polyprotein expression cassettes, single protein and multigene expression cassettes can also be integrated into the Tn7 attachment site (mini-attTn7) harbored by the LacZ (lacZα) gene, or the LoxP site of the MultiBac baculovirus. Ap stands for ampicillin. The F-Replicon is a single-copy bacterial origin of replication. For reagents contact: iberger@embl.fr

    Journal: Structural Genomics

    Article Title: Multiprotein Complex Production in Insect Cells by Using Polyproteins

    doi: 10.1007/978-1-62703-691-7_8

    Figure Lengend Snippet: Integration of polyprotein expression cassettes into MultiBac baculovirus. ( a ) MultiBac Acceptor vectors pPBac, pKL-PBac, and pOmni-PBac, tailored for polyprotein production, are shown schematically ( top ). They contain a regular ColE1 origin of replication and a polyprotein expression cassette, which encodes an N-terminal TEV protease and a C-terminal CFP spaced by a TEV cleavage site (tcs). BstEII and RsrII sites are used for inserting the polyprotein encoding ORF of interest. Donor vectors pIDC, pIDK, and pIDS contain a conditional origin of replication derived from the R6Kγ phage [ 13 ]. The multiplication module flanking the expression cassettes contain a homing endonuclease site and a complementary BstXI site ( boxes in light blue ). Polh and p10 are baculoviral very late promoters; SV40 and HSVtk are polyadenylation signals. MCS1 and MCS2 stand for multiple cloning sites. Tn7L and Tn7R are specific DNA sequences for Tn7 transposition; the lef2/603 and Ori1629 homology regions are shown as gray boxes . LoxP sites are shown as red balls . Cm stands for chloramphenicol, Gn for gentamicin, Kn for kanamycin, Sp for spectinomycin. ( b ) Besides polyprotein expression cassettes, single protein and multigene expression cassettes can also be integrated into the Tn7 attachment site (mini-attTn7) harbored by the LacZ (lacZα) gene, or the LoxP site of the MultiBac baculovirus. Ap stands for ampicillin. The F-Replicon is a single-copy bacterial origin of replication. For reagents contact: iberger@embl.fr

    Article Snippet: Materials for Inserting Polyprotein Constructs into Transfer Vectors via Restriction–Ligation Cloning Restriction endonucleases BstEII and RsrII and reaction buffers (New England Biolabs, NEB).

    Techniques: Expressing, Derivative Assay, Clone Assay