magnesium rna fragmentation module  (New England Biolabs)


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    NEBNext Magnesium RNA Fragmentation Module
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    NEBNext Magnesium RNA Fragmentation Module 200 rxns
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    e6150s
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    Category:
    mRNA Template Preparation for PCR
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    New England Biolabs magnesium rna fragmentation module
    NEBNext Magnesium RNA Fragmentation Module
    NEBNext Magnesium RNA Fragmentation Module 200 rxns
    https://www.bioz.com/result/magnesium rna fragmentation module/product/New England Biolabs
    Average 99 stars, based on 895 article reviews
    Price from $9.99 to $1999.99
    magnesium rna fragmentation module - by Bioz Stars, 2020-10
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    1) Product Images from "Landscape of RNA polyadenylation in E. coli"

    Article Title: Landscape of RNA polyadenylation in E. coli

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw894

    Scheme of the RNA preparation prior to the RNA-seq. ( A ) A hypothetical transcript and its degradation intermediates are illustrated. A mRNA with 5΄ triphosphate, stabilizing secondary structure near the 5΄-end and a terminator structure at the 3΄-end is shown. Three fragments with 5΄-monophosphate ends generated by endonucleolytic cleavages are also shown. Total RNA preparations from wt and pcnB mutant were incubated with RNA ligase and the PSS primer to tag 5΄-monophosphorylated RNAs. Excess PSS adaptors were eliminated and samples were incubated with TEX to remove untagged 5΄-monophosphorylated RNA molecules. RNA polyphosphatase was then used to eliminate γ and β-phosphates from primary transcripts, the TSS adaptor was ligated and TSS adaptor in excess eliminated. Total RNAs were then fragmented. Other extremities such as 5΄-hydroxyls generated by toxin cleavage, or RNA harboring a 5΄-NAD modification as a bacterial cap ( 66 , 67 ) will not have been tagged. ( B ) Differential expression profiles for the transcripts of the rpsO-pnp operon in the untagged (Int), PSS and TSS fractions from the wild-type (light grey) and pcnB deletion (dark grey). The arrow and the scissor refer to the rpsO transcription start and the RNase III upstream cleavage site, respectively. Expression level is indicated as reads/nt as a function of the gene's coordinates, which are shown under the RNA-seq profiles.
    Figure Legend Snippet: Scheme of the RNA preparation prior to the RNA-seq. ( A ) A hypothetical transcript and its degradation intermediates are illustrated. A mRNA with 5΄ triphosphate, stabilizing secondary structure near the 5΄-end and a terminator structure at the 3΄-end is shown. Three fragments with 5΄-monophosphate ends generated by endonucleolytic cleavages are also shown. Total RNA preparations from wt and pcnB mutant were incubated with RNA ligase and the PSS primer to tag 5΄-monophosphorylated RNAs. Excess PSS adaptors were eliminated and samples were incubated with TEX to remove untagged 5΄-monophosphorylated RNA molecules. RNA polyphosphatase was then used to eliminate γ and β-phosphates from primary transcripts, the TSS adaptor was ligated and TSS adaptor in excess eliminated. Total RNAs were then fragmented. Other extremities such as 5΄-hydroxyls generated by toxin cleavage, or RNA harboring a 5΄-NAD modification as a bacterial cap ( 66 , 67 ) will not have been tagged. ( B ) Differential expression profiles for the transcripts of the rpsO-pnp operon in the untagged (Int), PSS and TSS fractions from the wild-type (light grey) and pcnB deletion (dark grey). The arrow and the scissor refer to the rpsO transcription start and the RNase III upstream cleavage site, respectively. Expression level is indicated as reads/nt as a function of the gene's coordinates, which are shown under the RNA-seq profiles.

    Techniques Used: RNA Sequencing Assay, Generated, Mutagenesis, Incubation, Modification, Expressing

    Genome wide analysis of misregulated transcripts. The most abundant RNA fragments detected in the PSS fraction ( > 40 rpkm) accumulated in the mutant relative to the wt strain (log 2 FC > 1.5) were selected. The folding energy of these 129 RNAs was normalized relative to their length (normalized mfe) and presented as a function of the relative accumulation between the two strains (FC). Fragments derived from CDS are shown by red triangles and from REP sequences by green triangles. Randomly selected sequences (see Materials and Methods) were also folded and plotted as a function of their FC (blue triangles). Distributions of FC and normalized energy of the various populations are presented at the bottom and the right side of the graph respectively, with the same colors. Significantly different distributions from a random selection are indicated with one star ( P- value
    Figure Legend Snippet: Genome wide analysis of misregulated transcripts. The most abundant RNA fragments detected in the PSS fraction ( > 40 rpkm) accumulated in the mutant relative to the wt strain (log 2 FC > 1.5) were selected. The folding energy of these 129 RNAs was normalized relative to their length (normalized mfe) and presented as a function of the relative accumulation between the two strains (FC). Fragments derived from CDS are shown by red triangles and from REP sequences by green triangles. Randomly selected sequences (see Materials and Methods) were also folded and plotted as a function of their FC (blue triangles). Distributions of FC and normalized energy of the various populations are presented at the bottom and the right side of the graph respectively, with the same colors. Significantly different distributions from a random selection are indicated with one star ( P- value

    Techniques Used: Genome Wide, Mutagenesis, Derivative Assay, Selection

    2) Product Images from "Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX"

    Article Title: Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky932

    SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.
    Figure Legend Snippet: SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.

    Techniques Used: Footprinting, Labeling, Generated, Binding Assay

    In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.
    Figure Legend Snippet: In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.

    Techniques Used: Generated, Polyacrylamide Gel Electrophoresis

    3) Product Images from "Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction"

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-44457-z

    Saturation curves and differences in coverage for the 962 miRNAs in the Miltenyi miRXplore miRNA reference set for TGIRT-seq with or without different bias correction compared to published datasets for established small RNA-seq methods. For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. ( A ) RNA-seq saturation curves. The curves show the number of reference set miRNAs with at least 10 reads at bins of 200 reads. As additional reads were included, the number of miRNAs with at least 10 reads increased. Curves were truncated at 3 million reads. The dotted red line at the top indicates the number of miRNAs in the Miltenyi miRXplore reference set. Each curve represents combined datasets, color-coded by the sequencing method as shown in the Figure for the best (4N ligation/NEXTflex; n = 24) and worst (NEBNext; n = 12) methods from the comparison of Giraldez et al . 36 , as well as TGIRT-seq (n = 3 for libraries prepared with the NTT, MTT, and NTC adapters), TGIRT-seq with the NTTR adapter (n = 3), TGIRT-seq with the NTT adapter and an R1R adapter containing six randomized 5′-end positions (NTT/6N; n = 1), and the TGIRT-CircLigase method (n = 1; Mohr et al . 6 ). Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. ( B ) Violin plots of miRNA abundance in datasets obtained by different methods. The plots show the distribution of log 10 CPM for each miRNA in the reference set for each library preparation method (miRNA count = 2,886 for NTTc, 2,885 for NTCc, 23,088 for 4N ligation, 961 for TGIRT-CircLigase, 2,886 for NTTR, 5,522 for NEXTflex, 2,886 for MTT, 2,886 for NTC, 2,886 for NTT, 962 for NTT/6N, 30,757 for TruSeq, 3,815 for CleanTag, and 11,452 for NEBNext). NTTc and NTCc denote TGIRT-seq datasets obtained using the NTT or NTC adapters that were computationally corrected using the random forest regression model trained with the combined NTT datasets (Fig. 5C,D ). The black horizontal line indicates the expected CPM values (CPM = 1,039.5) for each miRNA for a uniform distribution of 1,000,000 reads to 962 miRNAs ( i . e ., unbiased sampling for each miRNA). The library preparation and correction methods are ordered from the lowest to highest deviation between the median CPM (white point within the violin) and the expected CPM. The black boxes in the violins indicate the interval between first and third quartiles, and the vertical lines indicate the 95% confidence interval for each method.
    Figure Legend Snippet: Saturation curves and differences in coverage for the 962 miRNAs in the Miltenyi miRXplore miRNA reference set for TGIRT-seq with or without different bias correction compared to published datasets for established small RNA-seq methods. For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. ( A ) RNA-seq saturation curves. The curves show the number of reference set miRNAs with at least 10 reads at bins of 200 reads. As additional reads were included, the number of miRNAs with at least 10 reads increased. Curves were truncated at 3 million reads. The dotted red line at the top indicates the number of miRNAs in the Miltenyi miRXplore reference set. Each curve represents combined datasets, color-coded by the sequencing method as shown in the Figure for the best (4N ligation/NEXTflex; n = 24) and worst (NEBNext; n = 12) methods from the comparison of Giraldez et al . 36 , as well as TGIRT-seq (n = 3 for libraries prepared with the NTT, MTT, and NTC adapters), TGIRT-seq with the NTTR adapter (n = 3), TGIRT-seq with the NTT adapter and an R1R adapter containing six randomized 5′-end positions (NTT/6N; n = 1), and the TGIRT-CircLigase method (n = 1; Mohr et al . 6 ). Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. ( B ) Violin plots of miRNA abundance in datasets obtained by different methods. The plots show the distribution of log 10 CPM for each miRNA in the reference set for each library preparation method (miRNA count = 2,886 for NTTc, 2,885 for NTCc, 23,088 for 4N ligation, 961 for TGIRT-CircLigase, 2,886 for NTTR, 5,522 for NEXTflex, 2,886 for MTT, 2,886 for NTC, 2,886 for NTT, 962 for NTT/6N, 30,757 for TruSeq, 3,815 for CleanTag, and 11,452 for NEBNext). NTTc and NTCc denote TGIRT-seq datasets obtained using the NTT or NTC adapters that were computationally corrected using the random forest regression model trained with the combined NTT datasets (Fig. 5C,D ). The black horizontal line indicates the expected CPM values (CPM = 1,039.5) for each miRNA for a uniform distribution of 1,000,000 reads to 962 miRNAs ( i . e ., unbiased sampling for each miRNA). The library preparation and correction methods are ordered from the lowest to highest deviation between the median CPM (white point within the violin) and the expected CPM. The black boxes in the violins indicate the interval between first and third quartiles, and the vertical lines indicate the 95% confidence interval for each method.

    Techniques Used: RNA Sequencing Assay, In Silico, Sequencing, Ligation, MTT Assay, Sampling

    4) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.282616.116

    DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.
    Figure Legend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

    Techniques Used: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection

    Analysis of cellular RNAs in DUSP11 knockout cell lines. ( A ) Host gene expression (RefSeq genes ≤500 nt with no annotated coding sequence) in HEK293T cells with or without Terminator treatment assayed by unfragmented TGIRT-seq. Read counts from the indicated cell line library mapping to annotated host genes in reads per million mapped (RPMM) are plotted on each axis. snoRNAs are indicated with blue circles. ( B ) Expression analysis of select host RNAP III transcribed genes in HEK293T, HEK293T DUSP11 knockout, A549, and A549 DUSP11 knockout cell lines assayed by fragmented TGIRT-seq with and without Terminator treatment. The log base 2 ratio of gene counts from the indicated libraries is plotted on each axis. ( C ) Northern blot analysis of candidate ncRNA DUSP11 targets from the indicated cell lines. EtBr-stained low-molecular-weight RNA is provided as an additional loading control. The membrane was first probed for vtRNA1-2, stripped, and reprobed for the indicated RNAs. ( D ) Model for the role of DUSP11 in RNA silencing and modulation of RNAP III transcripts in mammalian cells. RNAP III transcribed RNAs initially contain a 5′ triphosphate. DUSP11 dephosphorylates a fraction of these RNAs. This reduces the steady-state level and alters the activity/function of some RNAs. For RNAP III transcribed miRNA precursors, the 5p arm of the resulting 5′ monophosphorylated miRNA precursors is predominantly loaded into AGO proteins to generate stable/functional 5p RISCs. The miRNA precursors that remain 5′ triphosphorylated predominantly load the 3p miRNA in AGO, while the 5′ triphosphorylated 5p miRNAs are rapidly degraded. Furthermore, the 5′ triphosphorylated 5p miRNAs that are incorporated into AGO to generate an unstable 5p RISC, promoting degradation of the complex/5p miRNA. The red dashed arrow indicates the DUSP11-dependent, Dicer-independent route for the BLV 5p miRNAs and other noncanonical interfering RNAs.
    Figure Legend Snippet: Analysis of cellular RNAs in DUSP11 knockout cell lines. ( A ) Host gene expression (RefSeq genes ≤500 nt with no annotated coding sequence) in HEK293T cells with or without Terminator treatment assayed by unfragmented TGIRT-seq. Read counts from the indicated cell line library mapping to annotated host genes in reads per million mapped (RPMM) are plotted on each axis. snoRNAs are indicated with blue circles. ( B ) Expression analysis of select host RNAP III transcribed genes in HEK293T, HEK293T DUSP11 knockout, A549, and A549 DUSP11 knockout cell lines assayed by fragmented TGIRT-seq with and without Terminator treatment. The log base 2 ratio of gene counts from the indicated libraries is plotted on each axis. ( C ) Northern blot analysis of candidate ncRNA DUSP11 targets from the indicated cell lines. EtBr-stained low-molecular-weight RNA is provided as an additional loading control. The membrane was first probed for vtRNA1-2, stripped, and reprobed for the indicated RNAs. ( D ) Model for the role of DUSP11 in RNA silencing and modulation of RNAP III transcripts in mammalian cells. RNAP III transcribed RNAs initially contain a 5′ triphosphate. DUSP11 dephosphorylates a fraction of these RNAs. This reduces the steady-state level and alters the activity/function of some RNAs. For RNAP III transcribed miRNA precursors, the 5p arm of the resulting 5′ monophosphorylated miRNA precursors is predominantly loaded into AGO proteins to generate stable/functional 5p RISCs. The miRNA precursors that remain 5′ triphosphorylated predominantly load the 3p miRNA in AGO, while the 5′ triphosphorylated 5p miRNAs are rapidly degraded. Furthermore, the 5′ triphosphorylated 5p miRNAs that are incorporated into AGO to generate an unstable 5p RISC, promoting degradation of the complex/5p miRNA. The red dashed arrow indicates the DUSP11-dependent, Dicer-independent route for the BLV 5p miRNAs and other noncanonical interfering RNAs.

    Techniques Used: Knock-Out, Expressing, Sequencing, Northern Blot, Staining, Molecular Weight, Activity Assay, Functional Assay

    5) Product Images from "Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX"

    Article Title: Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky932

    SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.
    Figure Legend Snippet: SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.

    Techniques Used: Footprinting, Labeling, Generated, Binding Assay

    In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.
    Figure Legend Snippet: In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.

    Techniques Used: Generated, Polyacrylamide Gel Electrophoresis

    Related Articles

    Purification:

    Article Title: Sex-Dependent RNA Editing and N6-adenosine RNA Methylation Profiling in the Gonads of a Fish, the Olive Flounder (Paralichthys olivaceus)
    Article Snippet: .. Following purification, the poly (A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, United States) for 7 min under 86°C. .. Then the cleaved RNA fragments were incubated for 2 h at 4°C with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630).

    Article Title: Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX
    Article Snippet: .. To generate ladders, 1 μl of the purified RNA was separately subjected to hydrolysis using the Next Magnesium RNA Fragmentation module (–OH) or RNase T1 digestion (T1) (NEB). .. Reactions were resolved by 8% urea–PAGE, exposed on a phoshorimager screen, and scanned using the Storm 820 imaging system (GE Healthcare).

    Article Title: Landscape of RNA polyadenylation in E. coli
    Article Snippet: .. RNAs were then fragmented according to the NEBnext Magnesium RNA fragmentation module and purified on an RNEasy column (Qiagen). .. Purified fragmented molecules were incubated with antarctic alkaline phosphatase then T4 polynucleotide kinase (NEB).

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    New England Biolabs magnesium rna fragmentation module
    Scheme of the <t>RNA</t> preparation prior to the RNA-seq. ( A ) A hypothetical transcript and its degradation intermediates are illustrated. A mRNA with 5΄ triphosphate, stabilizing secondary structure near the 5΄-end and a terminator structure at the 3΄-end is shown. Three fragments with 5΄-monophosphate ends generated by endonucleolytic cleavages are also shown. Total RNA preparations from wt and pcnB mutant were incubated with RNA ligase and the PSS primer to tag 5΄-monophosphorylated <t>RNAs.</t> Excess PSS adaptors were eliminated and samples were incubated with TEX to remove untagged 5΄-monophosphorylated RNA molecules. RNA polyphosphatase was then used to eliminate γ and β-phosphates from primary transcripts, the TSS adaptor was ligated and TSS adaptor in excess eliminated. Total RNAs were then fragmented. Other extremities such as 5΄-hydroxyls generated by toxin cleavage, or RNA harboring a 5΄-NAD modification as a bacterial cap ( 66 , 67 ) will not have been tagged. ( B ) Differential expression profiles for the transcripts of the rpsO-pnp operon in the untagged (Int), PSS and TSS fractions from the wild-type (light grey) and pcnB deletion (dark grey). The arrow and the scissor refer to the rpsO transcription start and the RNase III upstream cleavage site, respectively. Expression level is indicated as reads/nt as a function of the gene's coordinates, which are shown under the RNA-seq profiles.
    Magnesium Rna Fragmentation Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/magnesium rna fragmentation module/product/New England Biolabs
    Average 99 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    magnesium rna fragmentation module - by Bioz Stars, 2020-10
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    Scheme of the RNA preparation prior to the RNA-seq. ( A ) A hypothetical transcript and its degradation intermediates are illustrated. A mRNA with 5΄ triphosphate, stabilizing secondary structure near the 5΄-end and a terminator structure at the 3΄-end is shown. Three fragments with 5΄-monophosphate ends generated by endonucleolytic cleavages are also shown. Total RNA preparations from wt and pcnB mutant were incubated with RNA ligase and the PSS primer to tag 5΄-monophosphorylated RNAs. Excess PSS adaptors were eliminated and samples were incubated with TEX to remove untagged 5΄-monophosphorylated RNA molecules. RNA polyphosphatase was then used to eliminate γ and β-phosphates from primary transcripts, the TSS adaptor was ligated and TSS adaptor in excess eliminated. Total RNAs were then fragmented. Other extremities such as 5΄-hydroxyls generated by toxin cleavage, or RNA harboring a 5΄-NAD modification as a bacterial cap ( 66 , 67 ) will not have been tagged. ( B ) Differential expression profiles for the transcripts of the rpsO-pnp operon in the untagged (Int), PSS and TSS fractions from the wild-type (light grey) and pcnB deletion (dark grey). The arrow and the scissor refer to the rpsO transcription start and the RNase III upstream cleavage site, respectively. Expression level is indicated as reads/nt as a function of the gene's coordinates, which are shown under the RNA-seq profiles.

    Journal: Nucleic Acids Research

    Article Title: Landscape of RNA polyadenylation in E. coli

    doi: 10.1093/nar/gkw894

    Figure Lengend Snippet: Scheme of the RNA preparation prior to the RNA-seq. ( A ) A hypothetical transcript and its degradation intermediates are illustrated. A mRNA with 5΄ triphosphate, stabilizing secondary structure near the 5΄-end and a terminator structure at the 3΄-end is shown. Three fragments with 5΄-monophosphate ends generated by endonucleolytic cleavages are also shown. Total RNA preparations from wt and pcnB mutant were incubated with RNA ligase and the PSS primer to tag 5΄-monophosphorylated RNAs. Excess PSS adaptors were eliminated and samples were incubated with TEX to remove untagged 5΄-monophosphorylated RNA molecules. RNA polyphosphatase was then used to eliminate γ and β-phosphates from primary transcripts, the TSS adaptor was ligated and TSS adaptor in excess eliminated. Total RNAs were then fragmented. Other extremities such as 5΄-hydroxyls generated by toxin cleavage, or RNA harboring a 5΄-NAD modification as a bacterial cap ( 66 , 67 ) will not have been tagged. ( B ) Differential expression profiles for the transcripts of the rpsO-pnp operon in the untagged (Int), PSS and TSS fractions from the wild-type (light grey) and pcnB deletion (dark grey). The arrow and the scissor refer to the rpsO transcription start and the RNase III upstream cleavage site, respectively. Expression level is indicated as reads/nt as a function of the gene's coordinates, which are shown under the RNA-seq profiles.

    Article Snippet: RNAs were then fragmented according to the NEBnext Magnesium RNA fragmentation module and purified on an RNEasy column (Qiagen).

    Techniques: RNA Sequencing Assay, Generated, Mutagenesis, Incubation, Modification, Expressing

    Genome wide analysis of misregulated transcripts. The most abundant RNA fragments detected in the PSS fraction ( > 40 rpkm) accumulated in the mutant relative to the wt strain (log 2 FC > 1.5) were selected. The folding energy of these 129 RNAs was normalized relative to their length (normalized mfe) and presented as a function of the relative accumulation between the two strains (FC). Fragments derived from CDS are shown by red triangles and from REP sequences by green triangles. Randomly selected sequences (see Materials and Methods) were also folded and plotted as a function of their FC (blue triangles). Distributions of FC and normalized energy of the various populations are presented at the bottom and the right side of the graph respectively, with the same colors. Significantly different distributions from a random selection are indicated with one star ( P- value

    Journal: Nucleic Acids Research

    Article Title: Landscape of RNA polyadenylation in E. coli

    doi: 10.1093/nar/gkw894

    Figure Lengend Snippet: Genome wide analysis of misregulated transcripts. The most abundant RNA fragments detected in the PSS fraction ( > 40 rpkm) accumulated in the mutant relative to the wt strain (log 2 FC > 1.5) were selected. The folding energy of these 129 RNAs was normalized relative to their length (normalized mfe) and presented as a function of the relative accumulation between the two strains (FC). Fragments derived from CDS are shown by red triangles and from REP sequences by green triangles. Randomly selected sequences (see Materials and Methods) were also folded and plotted as a function of their FC (blue triangles). Distributions of FC and normalized energy of the various populations are presented at the bottom and the right side of the graph respectively, with the same colors. Significantly different distributions from a random selection are indicated with one star ( P- value

    Article Snippet: RNAs were then fragmented according to the NEBnext Magnesium RNA fragmentation module and purified on an RNEasy column (Qiagen).

    Techniques: Genome Wide, Mutagenesis, Derivative Assay, Selection

    SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.

    Journal: Nucleic Acids Research

    Article Title: Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX

    doi: 10.1093/nar/gky932

    Figure Lengend Snippet: SOX binds to a stretch of adenosines upstream of the cleavage site. ( A ) An RNA footprinting assay was carried out by incubating 5′ 32 P-labeled LIMD1 -54 with RNase T1 in the presence (lanes 3–7) or absence (lane 2) of a dilution series of SOX (8–0.5 μM). Hydrolysis (–OH, lane 1) and RNase T1 (T1, lane 8) ladders of the RNA were also generated in order to map the location of protected sites. Lines on the right denote protected base pairs. ( B ) Diagram of LIMD1 -54 indicating sites protected from RNase T1 cleavage by SOX. The upstream SOX binding site is colored orange while the protected residues surrounding the cut site are shown in red.

    Article Snippet: To generate ladders, 1 μl of the purified RNA was separately subjected to hydrolysis using the Next Magnesium RNA Fragmentation module (–OH) or RNase T1 digestion (T1) (NEB).

    Techniques: Footprinting, Labeling, Generated, Binding Assay

    In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.

    Journal: Nucleic Acids Research

    Article Title: Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX

    doi: 10.1093/nar/gky932

    Figure Lengend Snippet: In-line structure probing of LIMD1 -54. ( A ) An in-line reaction (Rxn) was performed at room temperature for 24 h (lane 2) or 48 h (lane 3) at pH 8.3 to identify structured regions of the LIMD1 -54 RNA. Ladders were generated by subjecting the RNA to cleavage by RNase T1 (lane 8) or alkaline hydrolysis (–OH, lane 4–7). Products were separated by 8% urea PAGE, whereupon structured regions protected from cleavage were identified (green bars). No reaction (NR, lane1) refers to the input RNA. ( B ) Diagram showing the LIMD1 -54 structure as deduced from the in-line probing gel. Green color corresponds to the structured regions denoted by the bars in ( A ), while blue refers to unstructured regions.

    Article Snippet: To generate ladders, 1 μl of the purified RNA was separately subjected to hydrolysis using the Next Magnesium RNA Fragmentation module (–OH) or RNase T1 digestion (T1) (NEB).

    Techniques: Generated, Polyacrylamide Gel Electrophoresis

    Saturation curves and differences in coverage for the 962 miRNAs in the Miltenyi miRXplore miRNA reference set for TGIRT-seq with or without different bias correction compared to published datasets for established small RNA-seq methods. For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. ( A ) RNA-seq saturation curves. The curves show the number of reference set miRNAs with at least 10 reads at bins of 200 reads. As additional reads were included, the number of miRNAs with at least 10 reads increased. Curves were truncated at 3 million reads. The dotted red line at the top indicates the number of miRNAs in the Miltenyi miRXplore reference set. Each curve represents combined datasets, color-coded by the sequencing method as shown in the Figure for the best (4N ligation/NEXTflex; n = 24) and worst (NEBNext; n = 12) methods from the comparison of Giraldez et al . 36 , as well as TGIRT-seq (n = 3 for libraries prepared with the NTT, MTT, and NTC adapters), TGIRT-seq with the NTTR adapter (n = 3), TGIRT-seq with the NTT adapter and an R1R adapter containing six randomized 5′-end positions (NTT/6N; n = 1), and the TGIRT-CircLigase method (n = 1; Mohr et al . 6 ). Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. ( B ) Violin plots of miRNA abundance in datasets obtained by different methods. The plots show the distribution of log 10 CPM for each miRNA in the reference set for each library preparation method (miRNA count = 2,886 for NTTc, 2,885 for NTCc, 23,088 for 4N ligation, 961 for TGIRT-CircLigase, 2,886 for NTTR, 5,522 for NEXTflex, 2,886 for MTT, 2,886 for NTC, 2,886 for NTT, 962 for NTT/6N, 30,757 for TruSeq, 3,815 for CleanTag, and 11,452 for NEBNext). NTTc and NTCc denote TGIRT-seq datasets obtained using the NTT or NTC adapters that were computationally corrected using the random forest regression model trained with the combined NTT datasets (Fig. 5C,D ). The black horizontal line indicates the expected CPM values (CPM = 1,039.5) for each miRNA for a uniform distribution of 1,000,000 reads to 962 miRNAs ( i . e ., unbiased sampling for each miRNA). The library preparation and correction methods are ordered from the lowest to highest deviation between the median CPM (white point within the violin) and the expected CPM. The black boxes in the violins indicate the interval between first and third quartiles, and the vertical lines indicate the 95% confidence interval for each method.

    Journal: Scientific Reports

    Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

    doi: 10.1038/s41598-019-44457-z

    Figure Lengend Snippet: Saturation curves and differences in coverage for the 962 miRNAs in the Miltenyi miRXplore miRNA reference set for TGIRT-seq with or without different bias correction compared to published datasets for established small RNA-seq methods. For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. ( A ) RNA-seq saturation curves. The curves show the number of reference set miRNAs with at least 10 reads at bins of 200 reads. As additional reads were included, the number of miRNAs with at least 10 reads increased. Curves were truncated at 3 million reads. The dotted red line at the top indicates the number of miRNAs in the Miltenyi miRXplore reference set. Each curve represents combined datasets, color-coded by the sequencing method as shown in the Figure for the best (4N ligation/NEXTflex; n = 24) and worst (NEBNext; n = 12) methods from the comparison of Giraldez et al . 36 , as well as TGIRT-seq (n = 3 for libraries prepared with the NTT, MTT, and NTC adapters), TGIRT-seq with the NTTR adapter (n = 3), TGIRT-seq with the NTT adapter and an R1R adapter containing six randomized 5′-end positions (NTT/6N; n = 1), and the TGIRT-CircLigase method (n = 1; Mohr et al . 6 ). Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. ( B ) Violin plots of miRNA abundance in datasets obtained by different methods. The plots show the distribution of log 10 CPM for each miRNA in the reference set for each library preparation method (miRNA count = 2,886 for NTTc, 2,885 for NTCc, 23,088 for 4N ligation, 961 for TGIRT-CircLigase, 2,886 for NTTR, 5,522 for NEXTflex, 2,886 for MTT, 2,886 for NTC, 2,886 for NTT, 962 for NTT/6N, 30,757 for TruSeq, 3,815 for CleanTag, and 11,452 for NEBNext). NTTc and NTCc denote TGIRT-seq datasets obtained using the NTT or NTC adapters that were computationally corrected using the random forest regression model trained with the combined NTT datasets (Fig. 5C,D ). The black horizontal line indicates the expected CPM values (CPM = 1,039.5) for each miRNA for a uniform distribution of 1,000,000 reads to 962 miRNAs ( i . e ., unbiased sampling for each miRNA). The library preparation and correction methods are ordered from the lowest to highest deviation between the median CPM (white point within the violin) and the expected CPM. The black boxes in the violins indicate the interval between first and third quartiles, and the vertical lines indicate the 95% confidence interval for each method.

    Article Snippet: 2 μl of the resulting UHRR sample with ERCC spike-ins was ribo-depleted by using a Human/Mouse/Rat Ribo-zero rRNA removal kit (Illumina), fragmented to 70–100 nt by using an NEBNext Magnesium RNA Fragmentation Module (94 °C for 7 min; New England Biolabs), and treated with T4 polynucleotide kinase (Epicentre) to remove 3′ phosphates that impede TGIRT template-switching .

    Techniques: RNA Sequencing Assay, In Silico, Sequencing, Ligation, MTT Assay, Sampling

    DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

    Journal: Genes & Development

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    doi: 10.1101/gad.282616.116

    Figure Lengend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

    Article Snippet: Additionally, a subset of pretreated RNAs was fragmented prior to T4PNK treatment with an NEBNext Magnesium RNA fragmentation module (New England Biolabs) by incubating RNAs for 7 min at 94°C.

    Techniques: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection

    Analysis of cellular RNAs in DUSP11 knockout cell lines. ( A ) Host gene expression (RefSeq genes ≤500 nt with no annotated coding sequence) in HEK293T cells with or without Terminator treatment assayed by unfragmented TGIRT-seq. Read counts from the indicated cell line library mapping to annotated host genes in reads per million mapped (RPMM) are plotted on each axis. snoRNAs are indicated with blue circles. ( B ) Expression analysis of select host RNAP III transcribed genes in HEK293T, HEK293T DUSP11 knockout, A549, and A549 DUSP11 knockout cell lines assayed by fragmented TGIRT-seq with and without Terminator treatment. The log base 2 ratio of gene counts from the indicated libraries is plotted on each axis. ( C ) Northern blot analysis of candidate ncRNA DUSP11 targets from the indicated cell lines. EtBr-stained low-molecular-weight RNA is provided as an additional loading control. The membrane was first probed for vtRNA1-2, stripped, and reprobed for the indicated RNAs. ( D ) Model for the role of DUSP11 in RNA silencing and modulation of RNAP III transcripts in mammalian cells. RNAP III transcribed RNAs initially contain a 5′ triphosphate. DUSP11 dephosphorylates a fraction of these RNAs. This reduces the steady-state level and alters the activity/function of some RNAs. For RNAP III transcribed miRNA precursors, the 5p arm of the resulting 5′ monophosphorylated miRNA precursors is predominantly loaded into AGO proteins to generate stable/functional 5p RISCs. The miRNA precursors that remain 5′ triphosphorylated predominantly load the 3p miRNA in AGO, while the 5′ triphosphorylated 5p miRNAs are rapidly degraded. Furthermore, the 5′ triphosphorylated 5p miRNAs that are incorporated into AGO to generate an unstable 5p RISC, promoting degradation of the complex/5p miRNA. The red dashed arrow indicates the DUSP11-dependent, Dicer-independent route for the BLV 5p miRNAs and other noncanonical interfering RNAs.

    Journal: Genes & Development

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    doi: 10.1101/gad.282616.116

    Figure Lengend Snippet: Analysis of cellular RNAs in DUSP11 knockout cell lines. ( A ) Host gene expression (RefSeq genes ≤500 nt with no annotated coding sequence) in HEK293T cells with or without Terminator treatment assayed by unfragmented TGIRT-seq. Read counts from the indicated cell line library mapping to annotated host genes in reads per million mapped (RPMM) are plotted on each axis. snoRNAs are indicated with blue circles. ( B ) Expression analysis of select host RNAP III transcribed genes in HEK293T, HEK293T DUSP11 knockout, A549, and A549 DUSP11 knockout cell lines assayed by fragmented TGIRT-seq with and without Terminator treatment. The log base 2 ratio of gene counts from the indicated libraries is plotted on each axis. ( C ) Northern blot analysis of candidate ncRNA DUSP11 targets from the indicated cell lines. EtBr-stained low-molecular-weight RNA is provided as an additional loading control. The membrane was first probed for vtRNA1-2, stripped, and reprobed for the indicated RNAs. ( D ) Model for the role of DUSP11 in RNA silencing and modulation of RNAP III transcripts in mammalian cells. RNAP III transcribed RNAs initially contain a 5′ triphosphate. DUSP11 dephosphorylates a fraction of these RNAs. This reduces the steady-state level and alters the activity/function of some RNAs. For RNAP III transcribed miRNA precursors, the 5p arm of the resulting 5′ monophosphorylated miRNA precursors is predominantly loaded into AGO proteins to generate stable/functional 5p RISCs. The miRNA precursors that remain 5′ triphosphorylated predominantly load the 3p miRNA in AGO, while the 5′ triphosphorylated 5p miRNAs are rapidly degraded. Furthermore, the 5′ triphosphorylated 5p miRNAs that are incorporated into AGO to generate an unstable 5p RISC, promoting degradation of the complex/5p miRNA. The red dashed arrow indicates the DUSP11-dependent, Dicer-independent route for the BLV 5p miRNAs and other noncanonical interfering RNAs.

    Article Snippet: Additionally, a subset of pretreated RNAs was fragmented prior to T4PNK treatment with an NEBNext Magnesium RNA fragmentation module (New England Biolabs) by incubating RNAs for 7 min at 94°C.

    Techniques: Knock-Out, Expressing, Sequencing, Northern Blot, Staining, Molecular Weight, Activity Assay, Functional Assay