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    New England Biolabs mirna cloning linker
    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), <t>adenylated</t> (A)n, short genomically extended (g)n (
    Mirna Cloning Linker, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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