t4 rna ligase 2 truncated k227q (New England Biolabs)
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T4 RNA Ligase 2 truncated K227Q
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T4 RNA Ligase 2 truncated K227Q 10 000 units
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m0351l
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273
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10 000 units
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RNA Ligases
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New England Biolabs
t4 rna ligase 2 truncated k227q
T4 RNA Ligase 2 truncated K227Q 10 000 units
https://www.bioz.com/result/t4 rna ligase 2 truncated k227q/product/New England Biolabs
Average 90 stars, based on 18 article reviews
Price from $9.99 to $1999.99
T4 RNA Ligase 2 truncated K227Q 10 000 units
https://www.bioz.com/result/t4 rna ligase 2 truncated k227q/product/New England Biolabs
Average 90 stars, based on 18 article reviews
Price from $9.99 to $1999.99
t4 rna ligase 2 truncated k227q - by Bioz Stars,
2020-01
90/100 stars
Images
1) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"
Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
Journal: PLoS ONE
doi: 10.1371/journal.pone.0094619
Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis
Figure Legend 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.
Techniques Used: Ligation, Labeling
2) Product Images from "T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis"
Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis
Journal: BMC Biotechnology
doi: 10.1186/1472-6750-11-72
Figure Legend Snippet: Effect of PEG 8000 on ligase intermolecular strand-joining activity . Strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, ligase (13.8 pmol), and varying amounts of PEG 8000 for 1 hour at 25°C to assess the effect of PEG on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.
Techniques Used: Activity Assay, Labeling, Ligation, Binding Assay
Figure Legend Snippet: Deadenylation activity of T4 RNA ligase 2 truncated mutants . 5'-adenylated DNA adapters were incubated with an excess of ligase (13.8 pmol), and 12.5% PEG 8000 at 16°C overnight. Oligonucleotide substrates are depicted schematically above the gel. The contents of each sample were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold to visualize nucleic acid. Deadenylation of the DNA adapter (loss of 5'-App) is indicated by a band shift of ~1 nt towards the bottom of the gel. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
Techniques Used: Activity Assay, Incubation, Staining, Electrophoretic Mobility Shift Assay, Binding Assay
Figure Legend Snippet: Assaying the formation of side products by T4 RNA ligases . Intermolecular strand-joining reactions containing 5'-adenylated adapters, 21-mer 5'-PO 4 RNA acceptors, and ligase (1 pmol) were incubated at 16°C overnight in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. Products of the reaction were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Ladder = size standard ladder, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
Techniques Used: Incubation, Staining, Ligation, Binding Assay
![Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.](https://storage.googleapis.com/bioz_article_images/PMC3149579/1472-6750-11-72-9.jpg "Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were ...")
Figure Legend Snippet: Following AMP during ligation reactions with T4 RNA ligases . (A) 22-mer DNA adapters were 5'-adenylated with α- 32 P-labeled ATP (see materials and methods). Intermolecular strand-joining reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 21-mer 5'-PO 4 RNA acceptor, and ligase (1 pmol) were incubated overnight at 16°C in the presence of PEG 8000. Reaction products were resolved on a denaturing 15% acrylamide gel and radioactive molecules were visualized by exposure to Phosphor screens. The resulting products were either free AMP in solution (AMP*) or the adapter remaining adenylated (Ap*p-DNA). Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. (B) Determining the fate of AMP upon T4 RNA ligase-dependent deadenylation. Reactions containing radiolabeled DNA adapter (10 pmol) and ligase (14 pmol) were incubated overnight at 16°C in the presence of 12.5% PEG 8000. Oligonucleotide substrates are depicted schematically above the gel. P* denotes 32 P-phosphate. Reaction products were resolved and visualized as in (A). The resulting products were either free AMP in solution (AMP*), the adapter remaining adenylated (Ap*p-DNA), or AMP covalently bound to the ligase (AMP*-ligase). The lane labeled input contains only Ap*p-DNA. (C) Reactions identical to those in (B) were treated with Proteinase K prior to gel electrophoresis and detection. (D) Reactions containing 10 pmol radiolabeled DNA adapter, 5 pmol 28-mer [5'-PO 4 , 3'-blocked] RNA acceptor, and ligase (1 pmol) were incubated, resolved and detected as in (A). The resulting products were either free AMP in solution (AMP*), adenylated adapter (Ap*p-DNA), or Ap*p-28-mer RNA. The lane labeled RNA size control contains 5'- 32 PO 4 RNA, and the lane labeled input contains only Ap*p-DNA. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA. P* denotes 32 P-phosphate. In all panels, Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2 +MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP.
Techniques Used: Ligation, Labeling, Incubation, Acrylamide Gel Assay, Nucleic Acid Electrophoresis, Binding Assay
Figure Legend Snippet: Production of ligation side products by T4 RNA ligases . Intermolecular ligation reactions containing 5'-adenylated DNA adapters, 21-mer 5'-PO 4 RNA acceptors and ligase (1 pmol) were incubated at 16°C overnight with 12.5% PEG 8000. Products of the reactions were resolved on denaturing 15% acrylamide gels and stained with SYBR Gold. The bands corresponding to the input nucleic acids, the DNA adapter/RNA acceptor ligation product (39 bases), and larger side products are indicated. Rnl1 = T4 RNA ligase 1, Rnl2 = T4 RNA ligase 2, Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. Oligonucleotide substrates are depicted schematically above the gel. Grey lines represent RNA and black lines represent DNA.
Techniques Used: Ligation, Incubation, Staining, Binding Assay
Figure Legend Snippet: Purification and activity of T4 RNA Ligase 2 truncated mutants . (A) Aliquots of T4 RNA ligase 2 truncated and mutants were separated on 10-20% Tris-glycine SDS polyacrylamide gels and stained with Coomassie blue. The size (in kDa) of marker polypeptides are indicated on the left. (B) Intermolecular strand-joining activity of T4 RNA ligase 2 truncated mutants under multiple turnover conditions. 10 pmol 5'-adenylated 17-mer DNA was incubated for one hour at 25°C with 5 pmol 5'- FAM-labeled 31-mer RNA. 1 pmol of each ligase was added into reaction mixture. The reaction products were resolved on denaturing 15% acrylamide gels, scanned and quantified as described in the methods section. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments. * denotes difference in means p
Techniques Used: Purification, Activity Assay, Staining, Marker, Incubation, Labeling, Binding Assay
Figure Legend Snippet: Effect of pH on ligase intermolecular strand-joining activity . (A-D) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. (E-H) Intermolecular strand-joining reactions were carried out with 10 pmol 5'-adenylated 17-mer DNA, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (13.8 pmol) for 1 hour at 25°C to assess the effect of pH on ligation efficiency. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.
Techniques Used: Activity Assay, Labeling, Ligation, Binding Assay
Figure Legend Snippet: Analysis of intermolecular strand-joining over time . Strand-joining reactions were carried out with 10 pmol 5'-adenylated adapter, 5 pmol 31-mer 5'-FAM-labeled RNA acceptor, and ligase (1 pmol) over a span of 24 hours at 25°C to assess the progress of ligation reactions. Ligation efficiency was determined by resolving the material in the reactions on denaturing 15% acrylamide gels and quantifying the amount of ligation product versus input nucleic acid. Rnl2tr = T4 RNA ligase 2 truncated, Rnl2tr + MBP = T4 RNA ligase 2 truncated attached to an N-terminal maltose binding protein tag. All mutations indicated are substitutions in T4 Rnl2tr + MBP. Data are shown as the mean +/- SEM of at least three independent experiments.
Techniques Used: Labeling, Ligation, Binding Assay
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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 Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress Article Snippet: .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of Article Title: Analysis of genetically diverse macrophages reveals local and domain-wide mechanisms that control transcription factor binding and function Article Snippet: Here, prior to 3′Adapter ligation, samples were dissolved in 3.75 μl TET heated to 70°C for 2 minutes and placed on ice. .. Afterwards, to ligate the 3′ Illumina TruSeq adapter 10 μl of 3′MM was added [1 μl 10x T4 RNA ligase buffer, 6 μl 50% PEG8000, 1.5 μl ddH2 O+T, 0.25 μl heat-denaturated Illumina TruSeq 3′Adapter, 0.25 μl SUPERase-In, 1 μl Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe Article Snippet: .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1 Article Snippet: .. Alternatively, adapter pre-adenylation was performed using the 5′ DNA Adenylation Kit (New England Biolabs, Ipswich, MA) according to the manufacturer protocol. microRNA-Adapter Ligation. microRNA was ligated to the pre-adenylated dA adapters using Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Polymerase Chain Reaction:Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Protein Concentration:Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Fractions containing protein of interest were pooled and protein concentration was determined by a Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA) after overnight dialysis (100 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM DTT, 0.1 mM EDTA, 50% glycerol). .. T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, Sequencing:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses Article Snippet: .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: Paragraph title: Small RNA cDNA library preparation and sequencing ... RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Recombinant:Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress Article Snippet: Beads were subsequently incubated with 80 µl of 1xPNK buffer containing eight units of TSAP alkaline phosphatase (Promega) and 80 units of recombinant RNasin (Promega) for 1 h at 37 °C. .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of DC Protein Assay:Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Fractions containing protein of interest were pooled and protein concentration was determined by a Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA) after overnight dialysis (100 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM DTT, 0.1 mM EDTA, 50% glycerol). .. T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, RNA Sequencing Assay:Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses Article Snippet: Paragraph title: Library preparation for RNA-Seq ... The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by Magnetic Beads:Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses Article Snippet: .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by Mutagenesis:Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Paragraph title: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins ... T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Isolation:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: After DNase I treatment, the total RNA was isolated with TRIzol LS reagent (Invitrogen) and resuspended in 20 μL of water. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: RNA was isolated from 2.5 × 106 mating cells at the indicated time points post-mixing with the mirVana miRNA isolation kit (Ambion) or TRIzol reagent (Invitrogen). .. RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Labeling:Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress Article Snippet: After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of Purification:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: The RNAs were treated with DNase I again, and RNA was purified twice through P30 spin columns (Bio-Rad). .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of Article Title: Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway Article Snippet: Radiolabeled piRNAs from Aub and Piwi immunoprecipitates were resolved and purified with 8 M Urea 15% PAGE. .. Ligation was performed in the presence of 25% PEG 8000 ( ) with Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Paragraph title: Expression and purification of mutant T4 RNA ligase 2 truncated (Rnl2tr) proteins ... T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice Article Snippet: Briefly, rRNA was depleted from the purified monosomal RNA samples with RiboZero (Illumina, #MRZG12324). .. Recovered RNA was dephosphorylated with T4 Polynucleotide Kinase (NEB, #M0201S) and ligated with preadenylated adaptor in miRCat® -33 Conversion Oligos Pack (IDT) using Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: Two microliters of glycerol was added, the solution was incubated for another 10 min at room temperature in the dark, and RNAs were purified by ethanol precipitation. cDNA libraries of small RNAs were constructed as described ( ) with some modifications. .. RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Reverse Transcription Polymerase Chain Reaction:Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Immunoprecipitation:Article Title: Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants [W] ARGONAUTES Using Slicer-Defective Mutants [W] [OA] Article Snippet: Ten microliters (or 100%) of the immunoprecipitated RNA was mixed with 15 pmol of Cloning Linker 1 (IDT; see online) and incubated at 70°C for 2 min. .. The mixture was cooled on ice for 2 min before adding 1.5 μL of 10× T4 RNA ligase reaction buffer (NEB), 40 units of RNase OUT), and 10 units of De-Phosphorylation Assay:Article Title: Analysis of genetically diverse macrophages reveals local and domain-wide mechanisms that control transcription factor binding and function Article Snippet: RNA pellets were dissolved in 10 μl TET and a second round of dephosphorylation and BrdU enrichment was performed. .. Afterwards, to ligate the 3′ Illumina TruSeq adapter 10 μl of 3′MM was added [1 μl 10x T4 RNA ligase buffer, 6 μl 50% PEG8000, 1.5 μl ddH2 O+T, 0.25 μl heat-denaturated Illumina TruSeq 3′Adapter, 0.25 μl SUPERase-In, 1 μl Staining:Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Successful purification was monitored by SDS-PAGE followed by Coomassie blue staining (Life Technologies, Carlsbad, CA). .. T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, cDNA Library Assay:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: Paragraph title: Small RNA cDNA library preparation and sequencing ... RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or SDS Page:Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Successful purification was monitored by SDS-PAGE followed by Coomassie blue staining (Life Technologies, Carlsbad, CA). .. T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, Selection:Article Title: Analysis of genetically diverse macrophages reveals local and domain-wide mechanisms that control transcription factor binding and function Article Snippet: Afterwards, to ligate the 3′ Illumina TruSeq adapter 10 μl of 3′MM was added [1 μl 10x T4 RNA ligase buffer, 6 μl 50% PEG8000, 1.5 μl ddH2 O+T, 0.25 μl heat-denaturated Illumina TruSeq 3′Adapter, 0.25 μl SUPERase-In, 1 μl Agarose Gel Electrophoresis:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Ethanol Precipitation:Article Title: The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis Article Snippet: .. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris–HCl pH 7.5 and ligated in a 20 µL reaction overnight at 14°C to a preadenylated 3′-adaptor (5′-rATGGAATTCTCGGGTGCCAAGG-3′) using Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: Two microliters of glycerol was added, the solution was incubated for another 10 min at room temperature in the dark, and RNAs were purified by ethanol precipitation. cDNA libraries of small RNAs were constructed as described ( ) with some modifications. .. RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Concentration Assay:Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses Article Snippet: The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by Article Title: T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis Article Snippet: Elution was carried out by increasing the NaCl concentration by a gradient from 50 mM to 1 M over the course of 10 column volumes. .. T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 Truncated and, Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress Article Snippet: .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of Fractionation:Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena Article Snippet: RNA was recovered from the gel and ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′-App-TCGTATGCCGTCTTCTGCTTG-L-3′, where L is the 3′OH-blocking group; IDT) by T4 RNA ligase 2-truncated or Marker:Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, Lysis:Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe Article Snippet: After two washes with TN1000 buffer (100 mM Tris-HCl (pH 7.8), 2 M NaCl, 0.2% NP-40) and two washes with TN150 lysis buffer, the beads were incubated with GST-TEV protease for 2h at 16°C. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U Clear Native PAGE:Article Title: Preparation of Multiplexed Small RNA Libraries From Plants Article Snippet: 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, |