pre adenylated dna adapter  (New England Biolabs)


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    Name:
    Universal miRNA Cloning Linker
    Description:
    Universal miRNA Cloning Linker 5 ug
    Catalog Number:
    s1315s
    Price:
    151
    Size:
    5 ug
    Category:
    Probes and Primers
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    Structured Review

    New England Biolabs pre adenylated dna adapter
    Universal miRNA Cloning Linker
    Universal miRNA Cloning Linker 5 ug
    https://www.bioz.com/result/pre adenylated dna adapter/product/New England Biolabs
    Average 98 stars, based on 294 article reviews
    Price from $9.99 to $1999.99
    pre adenylated dna adapter - by Bioz Stars, 2020-08
    98/100 stars

    Images

    1) Product Images from "Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA"

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    Journal: RNA

    doi: 10.1261/rna.2242610

    Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.

    Techniques Used: Ligation, Staining, Incubation, Concentration Assay

    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Staining

    Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .
    Figure Legend Snippet: Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Incubation

    RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Methylation, Staining

    2) Product Images from "ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation"

    Article Title: ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

    Journal: Genes & Development

    doi: 10.1101/gad.326785.119

    ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended ( > 451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (
    Figure Legend Snippet: ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended ( > 451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (

    Techniques Used: CRISPR, Rapid Amplification of cDNA Ends, Sequencing

    Related Articles

    Clone Assay:

    Article Title: The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity
    Article Snippet: .. Samples were eluted with 8.5 uL nuclease free water and incubated with 1.5 uL of 0.5 ug/uL Universal miRNA Cloning Linker (NEB, catalog no. S1315S) and denatured at 80°C for 90 seconds. .. The denatured sample was then incubated with 1 uL T4 RNA Ligase 2, truncated (NEB, catalog no. M0242S), 2 uL 10× buffer, 1 uL SUPERase In, and 6 uL 50% PEG 8000 for 2.5 hours at room temperature.

    Article Title: Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication
    Article Snippet: .. A 5′-adenylated and 3′-blocked oligodeoxyribonucleotide microRNA (miRNA) cloning adaptor (5′ rApp CTG TAG GCA CCA TCA AT–NH2 3′; S1315; NEB) was ligated to the 3′ OH of the noncoding RNA using T4 RNA ligase 2 (M0351; NEB) by following the manufacturer's instructions. .. The cDNA was synthesized using a reverse primer (5′-ATT GAT GGT GCC TAC AG-3′) complementary to the adaptor sequence and MMLV reverse transcriptase (Invitrogen).

    Article Title: Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae
    Article Snippet: .. A universal miRNA cloning linker (NEB # S1315S) was ligated to the 3′-ends of the RNA using T4 RNA Ligase 2 (NEB #M0242 L) in the presence of PEG 8000 (2.5% w/v), DMSO and SUPERase-In at 37 °C for 2.5 h. The ligated products were resolved by electrophoresis on 15% TBE-Urea gel and the appropriate size fragments eluted from the gel. .. The linker-ligated RNA recovered from RPFs was directly subjected to reverse transcription, while that extracted from fragmented total RNA samples was used as input for the Ribozero reaction (Illumina Ribo-Zero Gold rRNA Removal Kit -Yeast) to remove rRNAs, and subsequently subjected to reverse transcription.

    Article Title: ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation
    Article Snippet: .. Briefly, DNase I-treated (Qiagen) total RNA was ligated to 5 µM pre-adenylated linker (Universal miRNA Cloning Linker, New England Bio Labs) with 280 U of T4 RNA ligase 2, truncated KO (New England BioLabs) in a 20-µL reaction at 25°C for 16 h with RNaseOUT and 25% PEG8000 (New England BioLabs). .. The ligation reaction was then cleaned using RNA Clean and Concentrator (Zymo Research) and cDNA was synthesized with a universal RT primer (5′-CTACGTAACGATTGATGGTGCCTACAG) using the SuperScript III reverse transcriptase (Thermo Fisher Scientific).

    Article Title: The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis
    Article Snippet: .. 3′-dephosphorylated RNA was ligated to a preadenylated miRNA cloning linker (IDT, Linker 1) using T4 RNl2(tr) (NEB) rather than enzymatically polyadenylated. .. Subtractive hybridization of rRNA contaminants was not performed.

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: .. Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes. .. We varied the amount of T4 Rnl2tr (New England Biolabs Inc.), ligation time, ligation temperature, and concentration of Polyethylene Glycol 8000 as indicated in the figures.

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1
    Article Snippet: .. 3′-end RNA mapping Mapping was performed according to the 3′-RNA ligase-mediated RACE method with minor modifications: Total RNA preparations were first 3′-dephosphorylated using T4 PNK for 1 h at 37 °C without ATP and pre-adenylated linker (Universal miRNA cloning linker, NEB) ligation was performed during 4 h at 22 °C in the presence of truncated RNA ligase 2 (NEB) . .. Reverse transcriptase reactions were performed using primer prE complementary to the linker sequence.

    Ligation:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: .. Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes. .. We varied the amount of T4 Rnl2tr (New England Biolabs Inc.), ligation time, ligation temperature, and concentration of Polyethylene Glycol 8000 as indicated in the figures.

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1
    Article Snippet: .. 3′-end RNA mapping Mapping was performed according to the 3′-RNA ligase-mediated RACE method with minor modifications: Total RNA preparations were first 3′-dephosphorylated using T4 PNK for 1 h at 37 °C without ATP and pre-adenylated linker (Universal miRNA cloning linker, NEB) ligation was performed during 4 h at 22 °C in the presence of truncated RNA ligase 2 (NEB) . .. Reverse transcriptase reactions were performed using primer prE complementary to the linker sequence.

    Purification:

    Article Title: Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation
    Article Snippet: .. Specific RNA bands were cut from the gel and eluted in 2× proteinase K buffer (100 mM Tris-HCl, pH 8.3; 25 mM EDTA, pH 8.0; 300 mM NaCl; 2% (w/v) SDS), purified and ligated to a miRNA universal linker (NEB) using T4 RNA ligase 1 in the absence of ATP. .. Ligation products were purified and reverse transcribed with Superscipt III (Invitrogen), and amplified using Titanium DNA polymerase (Clontech).

    Electrophoresis:

    Article Title: Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae
    Article Snippet: .. A universal miRNA cloning linker (NEB # S1315S) was ligated to the 3′-ends of the RNA using T4 RNA Ligase 2 (NEB #M0242 L) in the presence of PEG 8000 (2.5% w/v), DMSO and SUPERase-In at 37 °C for 2.5 h. The ligated products were resolved by electrophoresis on 15% TBE-Urea gel and the appropriate size fragments eluted from the gel. .. The linker-ligated RNA recovered from RPFs was directly subjected to reverse transcription, while that extracted from fragmented total RNA samples was used as input for the Ribozero reaction (Illumina Ribo-Zero Gold rRNA Removal Kit -Yeast) to remove rRNAs, and subsequently subjected to reverse transcription.

    Incubation:

    Article Title: The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity
    Article Snippet: .. Samples were eluted with 8.5 uL nuclease free water and incubated with 1.5 uL of 0.5 ug/uL Universal miRNA Cloning Linker (NEB, catalog no. S1315S) and denatured at 80°C for 90 seconds. .. The denatured sample was then incubated with 1 uL T4 RNA Ligase 2, truncated (NEB, catalog no. M0242S), 2 uL 10× buffer, 1 uL SUPERase In, and 6 uL 50% PEG 8000 for 2.5 hours at room temperature.

    CTG Assay:

    Article Title: Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication
    Article Snippet: .. A 5′-adenylated and 3′-blocked oligodeoxyribonucleotide microRNA (miRNA) cloning adaptor (5′ rApp CTG TAG GCA CCA TCA AT–NH2 3′; S1315; NEB) was ligated to the 3′ OH of the noncoding RNA using T4 RNA ligase 2 (M0351; NEB) by following the manufacturer's instructions. .. The cDNA was synthesized using a reverse primer (5′-ATT GAT GGT GCC TAC AG-3′) complementary to the adaptor sequence and MMLV reverse transcriptase (Invitrogen).

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    New England Biolabs pre adenylated dna adapter
    Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to <t>pre-adenylated</t> <t>DNA</t> adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.
    Pre Adenylated Dna Adapter, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pre adenylated dna adapter/product/New England Biolabs
    Average 98 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    pre adenylated dna adapter - by Bioz Stars, 2020-08
    98/100 stars
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    Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.

    Article Snippet: Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes.

    Techniques: Ligation, Staining, Incubation, Concentration Assay

    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Article Snippet: Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes.

    Techniques: Ligation, Staining

    Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .

    Article Snippet: Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes.

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Incubation

    RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Journal: RNA

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    doi: 10.1261/rna.2242610

    Figure Lengend Snippet: RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Article Snippet: Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes.

    Techniques: Ligation, Methylation, Staining

    ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended ( > 451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (

    Journal: Genes & Development

    Article Title: ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

    doi: 10.1101/gad.326785.119

    Figure Lengend Snippet: ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended ( > 451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (

    Article Snippet: Briefly, DNase I-treated (Qiagen) total RNA was ligated to 5 µM pre-adenylated linker (Universal miRNA Cloning Linker, New England Bio Labs) with 280 U of T4 RNA ligase 2, truncated KO (New England BioLabs) in a 20-µL reaction at 25°C for 16 h with RNaseOUT and 25% PEG8000 (New England BioLabs).

    Techniques: CRISPR, Rapid Amplification of cDNA Ends, Sequencing