rna  (New England Biolabs)


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    Name:
    NEBNext Magnesium RNA Fragmentation Module
    Description:
    NEBNext Magnesium RNA Fragmentation Module 200 rxns
    Catalog Number:
    e6150s
    Price:
    46
    Size:
    200 rxns
    Category:
    mRNA Template Preparation for PCR
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    Structured Review

    New England Biolabs rna
    NEBNext Magnesium RNA Fragmentation Module
    NEBNext Magnesium RNA Fragmentation Module 200 rxns
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 95 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Amplification:

    Article Title: Aire controls gene expression in the thymic epithelium with ordered stochasticity
    Article Snippet: .. First the fragmentation of the amplified RNA (aRNA) was performed using the Magnesium RNA Fragmentation Module (NEBNext E6150S). ..

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: Paragraph title: Linear RNA Amplification, Illumina Library Prep, and Sequencing ... Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ).

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Briefly, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064–014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012). .. Samples were again reverse-transcribed using a 3′ adaptor-specific primer and amplified using indexed Illumina RNA PCR primers (Illumina, RS-200-0012).

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. In summary, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064‐014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    Article Title: Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity
    Article Snippet: Paragraph title: Linear RNA amplification ... Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps.

    Synthesized:

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Briefly, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064–014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. In summary, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064‐014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    Incubation:

    Article Title: Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish
    Article Snippet: Purified mRNA was fragmented for 4 min at 94 °C using the NEBNext Magnesium RNA Fragmentation Module (NEB – E6150S) to approximately 200–500 bases, recovered by ethanol precipitation, and dissolved in 50 μl RNase-free water. .. Following a 3 h incubation at room temperature in the dark, excess biotin was removed by a chloroform/isoamyl alcohol (24:1) extraction as described [ ].

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: The aqueous phase was add to 0.5 mL of isopropyl alcohol, incubated for 10 min at room temperature, and centrifuged at 12,000 g for 10 min at 4°C. .. For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water.

    Article Title: Aire controls gene expression in the thymic epithelium with ordered stochasticity
    Article Snippet: 10.4 µL of a mix containing 1.6µL of ATP (75mM stock), 1.6µL of UTP (75mM stock), 1.6µL of GTP (75mM stock), 1.6µL of CTP (75mM stock), 1.6µL of T7 10X Reaction Buffer, 1.6µL of T7 Enzyme Mix and 0.8µL of RNAseOut was added to each 6µL cDNA pool, and incubated at 37°C lid 70°C for 14h. .. First the fragmentation of the amplified RNA (aRNA) was performed using the Magnesium RNA Fragmentation Module (NEBNext E6150S).

    Article Title: BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing
    Article Snippet: .. RNA fragmentation was done using the NEBNext Magnesium RNA Fragmentation Module (NEB, #E6150S) with incubation time at 94 °C for 1 or 2 min. ..

    Article Title: N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling
    Article Snippet: Sodium chloride was added, and polyadenylated RNA captured on oligo-dT-covered polystyrene beads during a 10-min incubation. .. The RNA was subjected to 100bases fragmentation using NEB fragmentation buffer (#E6150S, NEB, Ipswich, MA, USA) for 5 min.

    Modification:

    Article Title: Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection
    Article Snippet: About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega). .. The end-repaired RNAs were used to prepare the strand specific transcriptome, using the Small RNA Sample Preparation kit (E6160, NEB) according to the manufacturer protocol with some minor modification, including the SR Primer F3 being replaced with barcode primers.

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: Single-cell RNA sequencing Massively parallel scRNA-Seq of human bronchial airway cells was performed using a modified version of the CEL-Seq RNA library preparation protocol ( ). .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Ligation:

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5′ and 3′ ends of the RNA available for adapter ligation.

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5′ and 3′ ends of the RNA available for adapter ligation.

    Generated:

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: Complementary DNA generated from each of the 96 wells per plate was pooled, subjected to second-strand synthesis (Thermo Fisher, AM1751), and amplified by in vitro transcription (Thermo Fisher, AM1751). .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Sequencing:

    Article Title: Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection
    Article Snippet: Paragraph title: DNA and RNA library preparation, emulsion PCR and sequencing ... About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega).

    Article Title: A metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from 18 species
    Article Snippet: Paragraph title: Transcriptome Sequencing of H. monstrosus and R. aegyptiacus. ... The mRNA was fragmented using the NEBNext Mg Fragmentation module (NEB; E6150S) and column purified using RNeasy MinElute kit (Qiagen; 74204). mRNA ends were repaired using T4 Polynucleotide kinase (NEB; M0201L) and ATP (Ambion; AM8110G).

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: Paragraph title: Linear RNA Amplification, Illumina Library Prep, and Sequencing ... Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ).

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: .. Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5′ and 3′ ends of the RNA available for adapter ligation.

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: For each of the 12 recruited donors, one frozen 96-well PCR plate containing sorted cells was thawed on ice, and RNA was directly reverse-transcribed (Thermo Fisher, AM1751) from whole-cell lysate using primers composed of an anchored poly(dT), the 5′ Illumina adaptor sequence, a six-nucleotide well-specific barcode, a five-nucleotide unique molecular identifier (UMI), and a T7 RNA polymerase promoter. .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Libraries for RNA‐Seq were prepared following directional RNA‐Seq library preparation and sequencing. .. Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB).

    Article Title: Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity
    Article Snippet: These two rounds of linearly amplified aRNA products now carried the 3′-end of the polyA transcripts for mapping to coding regions plus the sample barcode to indicate which PCP it came from, UMI sequence for counting unique cell-endogenous parent mRNA molecules and one of the flanking sequence (RA5 adapter) for Illumina sequencing. .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps.

    RNA Sequencing Assay:

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: .. Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5′ and 3′ ends of the RNA available for adapter ligation.

    Article Title: Applying thiouracil (TU)-tagging for mouse transcriptome analysis
    Article Snippet: Isopropyl alcohol (Macron Fine Chemicals, cat. no. MK303202) Ethanol (EtOH) (Pharmco Aaper, cat. no. 11100020G) RNase-free water (H2 O) (Ambion cat. no. AM9938) 1M Tris pH 8.0 RNase-free (Ambion cat. no. AM9855G) 0.5M EDTA pH 8.0 RNase-free (Ambion cat. no. AM9260G) TURBO DNase (Invitrogen cat. no. AM2238) RNeasy Mini Kit (Qiagen cat. no. 74101) Qubit RNA BR Assay Kit (Invitrogen, cat. no. ) Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. ) Qubit assay tubes (Invitrogen, cat. no. ) NEBNext magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) Ribo-Zero Magnetic Kit (Epicentre, cat. no. MRZH11124) N,N-Dimethylformamide (Sigma, cat. no. D4551) EZ-Link Biotin-HDPD (Thermo Scientific, cat. no. 21341) μMacs Streptavidin Kit (Miltenyi Biotec, cat. no. 130-074-101) 2-mercaptoethanol (Sigma, cat. no. M3148) !CAUTION 2-mercaptoethanol is toxic and should only be used in a fume hood. .. Agencourt AMPure XP beads (Beckman Coulter, cat. no. ) RNeasy MinElute (Qiagen, cat. no. 74204) MinElute Reaction Cleanup Kit (Qiagen, cat. no. 28204) Tween-20 (Sigma, cat. no. P7949) ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, cat. no. SSV21106) FailSafe PCR Enzyme Mix (Epicentre, cat. no. FSE51100) ScriptSeq Index PCR Primers (Epicentre, cat. no. RSBC10948 (set 1))

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: Paragraph title: Single-cell RNA sequencing ... Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Paragraph title: RNA extraction and library preparation for RNA‐Seq ... Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB).

    Article Title: Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy
    Article Snippet: Paragraph title: RNA-Seq. ... After rRNA removal, the sample RNAs were fragmented via alkaline hydrolysis using the NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, E6150S).

    RNA HS Assay:

    Article Title: BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing
    Article Snippet: The RNA concentration was determined using the Qubit RNA HS Assay Kit (Invitrogen, #Q32852), and integrity was assessed using a Fragment Analyzer (Advanced Analytical). .. RNA fragmentation was done using the NEBNext Magnesium RNA Fragmentation Module (NEB, #E6150S) with incubation time at 94 °C for 1 or 2 min.

    Magnetic Beads:

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: For isolation of poly(A) RNA, total RNA was subjected to two rounds of purification using oligo(dT)-coupled magnetic beads according to the manufacturer’s instructions (Ambion). .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Isolation:

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water. .. Biotinylated RNA was then isolated from nonlabeled RNA using a μMacs streptavidin kit (Miltenyi Biotec, #130-074-101) following the manufacturer's directions with the exception of elution from the column in 100 mM β-mercaptoethanol.

    Article Title: A metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from 18 species
    Article Snippet: RNA was isolated from the immortalized cell lines HypLu/45.1 and HypNi/1.1 (H. monstrosus ) and RoNi/7.1 (R. aegyptiacus ) ( ) using the AllPrep DNA/RNA mini kit (Qiagen- 80204). mRNA isolation, clean up, and library prep was done by the Genome Sequencing and Analysis Facility at University of Texas at Austin. .. The mRNA was fragmented using the NEBNext Mg Fragmentation module (NEB; E6150S) and column purified using RNeasy MinElute kit (Qiagen; 74204). mRNA ends were repaired using T4 Polynucleotide kinase (NEB; M0201L) and ATP (Ambion; AM8110G).

    Article Title: BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing
    Article Snippet: RNA samples for library preparation Total RNA was isolated using TRI Reagent (Molecular Research Center, #TR118) followed by double precipitation with ethanol. .. RNA fragmentation was done using the NEBNext Magnesium RNA Fragmentation Module (NEB, #E6150S) with incubation time at 94 °C for 1 or 2 min.

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: For isolation of poly(A) RNA, total RNA was subjected to two rounds of purification using oligo(dT)-coupled magnetic beads according to the manufacturer’s instructions (Ambion). .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Article Title: N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling
    Article Snippet: RNA Preparation for RIP-Sequencing PolyA-RNA was isolated using the Genelute Direct RNA kit (#DMN10, Sigma). .. The RNA was subjected to 100bases fragmentation using NEB fragmentation buffer (#E6150S, NEB, Ipswich, MA, USA) for 5 min.

    Size-exclusion Chromatography:

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: Chloroform ( 200 μL) was added to the mix, and tubes were vortexed for 15 sec, incubated for 2–3 min at room temperature, and centrifugedat 12,000 g for 15 min at 4°C . .. For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water.

    Labeling:

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water. .. Remaining RNA (18 μL) was biotinylated using 25 μL of EZ-Link Biotin HPDP (Pierce Biotechnology, #21341) dissolved to 1 mg/mL in N,N-dimethylformamide in total reaction volume of 100 μL of 1× TE (pH 8.0) for 3 h. The labeled RNA was purified by RNeasy minikit and eluted in 20 μL of RNase-free water.

    Mouse Assay:

    Article Title: Applying thiouracil (TU)-tagging for mouse transcriptome analysis
    Article Snippet: Transgenic tissue-specific Cre recombinase mice (e.g., Tie2:Cre ( B6.Cg-Tg(Tek-cre)1Ywa/J ): The Jackson Laboratory, stock no. 008863) Transgenic CA > GFPstop > HA-UPRT mice (The Jackson Laboratory, stock no. 021469) !CAUTION All mouse procedures must be authorized by your institutional animal care and use committee in compliance with institutional and national animal care guidelines DMSO (Sigma, cat. no. D1435) Corn oil (Sigma, cat. no. .. Isopropyl alcohol (Macron Fine Chemicals, cat. no. MK303202) Ethanol (EtOH) (Pharmco Aaper, cat. no. 11100020G) RNase-free water (H2 O) (Ambion cat. no. AM9938) 1M Tris pH 8.0 RNase-free (Ambion cat. no. AM9855G) 0.5M EDTA pH 8.0 RNase-free (Ambion cat. no. AM9260G) TURBO DNase (Invitrogen cat. no. AM2238) RNeasy Mini Kit (Qiagen cat. no. 74101) Qubit RNA BR Assay Kit (Invitrogen, cat. no. ) Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. ) Qubit assay tubes (Invitrogen, cat. no. ) NEBNext magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) Ribo-Zero Magnetic Kit (Epicentre, cat. no. MRZH11124) N,N-Dimethylformamide (Sigma, cat. no. D4551) EZ-Link Biotin-HDPD (Thermo Scientific, cat. no. 21341) μMacs Streptavidin Kit (Miltenyi Biotec, cat. no. 130-074-101) 2-mercaptoethanol (Sigma, cat. no. M3148) !CAUTION 2-mercaptoethanol is toxic and should only be used in a fume hood.

    Polymerase Chain Reaction:

    Article Title: Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection
    Article Snippet: Paragraph title: DNA and RNA library preparation, emulsion PCR and sequencing ... About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega).

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Briefly, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064–014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    Article Title: Applying thiouracil (TU)-tagging for mouse transcriptome analysis
    Article Snippet: Isopropyl alcohol (Macron Fine Chemicals, cat. no. MK303202) Ethanol (EtOH) (Pharmco Aaper, cat. no. 11100020G) RNase-free water (H2 O) (Ambion cat. no. AM9938) 1M Tris pH 8.0 RNase-free (Ambion cat. no. AM9855G) 0.5M EDTA pH 8.0 RNase-free (Ambion cat. no. AM9260G) TURBO DNase (Invitrogen cat. no. AM2238) RNeasy Mini Kit (Qiagen cat. no. 74101) Qubit RNA BR Assay Kit (Invitrogen, cat. no. ) Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. ) Qubit assay tubes (Invitrogen, cat. no. ) NEBNext magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) Ribo-Zero Magnetic Kit (Epicentre, cat. no. MRZH11124) N,N-Dimethylformamide (Sigma, cat. no. D4551) EZ-Link Biotin-HDPD (Thermo Scientific, cat. no. 21341) μMacs Streptavidin Kit (Miltenyi Biotec, cat. no. 130-074-101) 2-mercaptoethanol (Sigma, cat. no. M3148) !CAUTION 2-mercaptoethanol is toxic and should only be used in a fume hood. .. Agencourt AMPure XP beads (Beckman Coulter, cat. no. ) RNeasy MinElute (Qiagen, cat. no. 74204) MinElute Reaction Cleanup Kit (Qiagen, cat. no. 28204) Tween-20 (Sigma, cat. no. P7949) ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, cat. no. SSV21106) FailSafe PCR Enzyme Mix (Epicentre, cat. no. FSE51100) ScriptSeq Index PCR Primers (Epicentre, cat. no. RSBC10948 (set 1))

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: For each of the 12 recruited donors, one frozen 96-well PCR plate containing sorted cells was thawed on ice, and RNA was directly reverse-transcribed (Thermo Fisher, AM1751) from whole-cell lysate using primers composed of an anchored poly(dT), the 5′ Illumina adaptor sequence, a six-nucleotide well-specific barcode, a five-nucleotide unique molecular identifier (UMI), and a T7 RNA polymerase promoter. .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. In summary, 3′ adapters and subsequently 5′ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064‐014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3′ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit.

    cDNA Library Assay:

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ). .. The resulting library was paired-end sequenced for 101 bp in Illumina HiSeq.

    Article Title: Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps. .. cDNA library was generated using Illumina TruSeq small RNA kit (Cat#RS-200-0012) and only 3′-adapter (RA3) need to be ligated enzymatically using truncated T4 RNA ligase (NEB M0242) on to the fragmented aRNA and the 5′ ligation step for RA5-adapter was skipped ( ).

    Agarose Gel Electrophoresis:

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: Then, mRNA concentration was measured by Qubit 2.0 (Invitrogen) and the integrity was tested by agarose gel electrophoresis and Agilent 2100 bioanalyzer. .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (CatE6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ). .. The resulting library was paired-end sequenced for 101 bp in Illumina HiSeq.

    Article Title: Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (CatE6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps. .. cDNA library was generated using Illumina TruSeq small RNA kit (Cat#RS-200-0012) and only 3′-adapter (RA3) need to be ligated enzymatically using truncated T4 RNA ligase (NEB M0242) on to the fragmented aRNA and the 5′ ligation step for RA5-adapter was skipped ( ).

    Purification:

    Article Title: Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish
    Article Snippet: .. Purified mRNA was fragmented for 4 min at 94 °C using the NEBNext Magnesium RNA Fragmentation Module (NEB – E6150S) to approximately 200–500 bases, recovered by ethanol precipitation, and dissolved in 50 μl RNase-free water. .. TU-tagged RNA was biotinylated using EZ-Link HPDP-Biotin (Thermo Scientific – 21341) by the addition of 25 μl 4x TE and 25 μl 1 mg/ml EZ-Link (dissolved in DMF) to the 50 μl RNA.

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: .. For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water. .. The RNA concentration was determined by Qubit, and a 2-μL sample was reserved as the total RNA control.

    Article Title: Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection
    Article Snippet: .. About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega). .. The end-repaired RNAs were used to prepare the strand specific transcriptome, using the Small RNA Sample Preparation kit (E6160, NEB) according to the manufacturer protocol with some minor modification, including the SR Primer F3 being replaced with barcode primers.

    Article Title: Aire controls gene expression in the thymic epithelium with ordered stochasticity
    Article Snippet: A second bead purification was performed similarly with 1x volume of RNA AMPure XP Beads and eluted in 6µL of RNase-free water. .. First the fragmentation of the amplified RNA (aRNA) was performed using the Magnesium RNA Fragmentation Module (NEBNext E6150S).

    Article Title: A metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from 18 species
    Article Snippet: .. The mRNA was fragmented using the NEBNext Mg Fragmentation module (NEB; E6150S) and column purified using RNeasy MinElute kit (Qiagen; 74204). mRNA ends were repaired using T4 Polynucleotide kinase (NEB; M0201L) and ATP (Ambion; AM8110G). ..

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ). .. The resulting library was paired-end sequenced for 101 bp in Illumina HiSeq.

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: For isolation of poly(A) RNA, total RNA was subjected to two rounds of purification using oligo(dT)-coupled magnetic beads according to the manufacturer’s instructions (Ambion). .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Article Title: N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling
    Article Snippet: After three washes in a spin column, purified mRNA was eluted in 100 μL of 10 mM Tris-HCl, pH 7.4. .. The RNA was subjected to 100bases fragmentation using NEB fragmentation buffer (#E6150S, NEB, Ipswich, MA, USA) for 5 min.

    Article Title: Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity
    Article Snippet: .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps. .. cDNA library was generated using Illumina TruSeq small RNA kit (Cat#RS-200-0012) and only 3′-adapter (RA3) need to be ligated enzymatically using truncated T4 RNA ligase (NEB M0242) on to the fragmented aRNA and the 5′ ligation step for RA5-adapter was skipped ( ).

    Article Title: Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy
    Article Snippet: Total RNA was purified from the cell lysates using TRIzol LS. .. After rRNA removal, the sample RNAs were fragmented via alkaline hydrolysis using the NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, E6150S).

    RNA Extraction:

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Paragraph title: RNA extraction and library preparation for RNA‐Seq ... Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB).

    Sample Prep:

    Article Title: Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection
    Article Snippet: About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega). .. The end-repaired RNAs were used to prepare the strand specific transcriptome, using the Small RNA Sample Preparation kit (E6160, NEB) according to the manufacturer protocol with some minor modification, including the SR Primer F3 being replaced with barcode primers.

    Article Title: Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
    Article Snippet: Directional RNA-seq library preparation and sequencing One μg of total RNA was fragmented to around 100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer’s protocol.

    Article Title: Unraveling the hidden universe of small proteins in bacterial genomes
    Article Snippet: Briefly, 1 μg of total RNA was fragmented into ~100–150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). .. Samples were further processed using the TruSeq Small RNA Sample Prep Kit (ref. RS‐200‐0012, Illumina) according to the manufacturer's protocol.

    In Vitro:

    Article Title: Aire controls gene expression in the thymic epithelium with ordered stochasticity
    Article Snippet: In vitro transcription was then conducted using the MEGAshortscript T7 transcription kit (Ambion AM1354). .. First the fragmentation of the amplified RNA (aRNA) was performed using the Magnesium RNA Fragmentation Module (NEBNext E6150S).

    Article Title: Mouse Cntnap2 and Human CNTNAP2 ASD Alleles Cell Autonomously Regulate PV+ Cortical Interneurons
    Article Snippet: RNA was linearly amplified by T7 RNA polymerase using 2 rounds of in vitro transcription (MessageAmp-II kit Life Technologies) according to the manufacturer’s recommended protocol with some modifications to make aRNA. .. Second round aRNAs were fragmented chemically using NEBNext® Magnesium RNA Fragmentation Module (Cat#E6150S), column purified using RNA MinElute (Qiagen) for final Illumina cDNA library preparation steps using Illumina TruSeq small RNA kit (Cat#RS-200-0012) ( ).

    Article Title: Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution
    Article Snippet: Complementary DNA generated from each of the 96 wells per plate was pooled, subjected to second-strand synthesis (Thermo Fisher, AM1751), and amplified by in vitro transcription (Thermo Fisher, AM1751). .. Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3′ adapter (Illumina, RS-200-0012).

    Transgenic Assay:

    Article Title: Applying thiouracil (TU)-tagging for mouse transcriptome analysis
    Article Snippet: Transgenic tissue-specific Cre recombinase mice (e.g., Tie2:Cre ( B6.Cg-Tg(Tek-cre)1Ywa/J ): The Jackson Laboratory, stock no. 008863) Transgenic CA > GFPstop > HA-UPRT mice (The Jackson Laboratory, stock no. 021469) !CAUTION All mouse procedures must be authorized by your institutional animal care and use committee in compliance with institutional and national animal care guidelines DMSO (Sigma, cat. no. D1435) Corn oil (Sigma, cat. no. .. Isopropyl alcohol (Macron Fine Chemicals, cat. no. MK303202) Ethanol (EtOH) (Pharmco Aaper, cat. no. 11100020G) RNase-free water (H2 O) (Ambion cat. no. AM9938) 1M Tris pH 8.0 RNase-free (Ambion cat. no. AM9855G) 0.5M EDTA pH 8.0 RNase-free (Ambion cat. no. AM9260G) TURBO DNase (Invitrogen cat. no. AM2238) RNeasy Mini Kit (Qiagen cat. no. 74101) Qubit RNA BR Assay Kit (Invitrogen, cat. no. ) Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. ) Qubit assay tubes (Invitrogen, cat. no. ) NEBNext magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) Ribo-Zero Magnetic Kit (Epicentre, cat. no. MRZH11124) N,N-Dimethylformamide (Sigma, cat. no. D4551) EZ-Link Biotin-HDPD (Thermo Scientific, cat. no. 21341) μMacs Streptavidin Kit (Miltenyi Biotec, cat. no. 130-074-101) 2-mercaptoethanol (Sigma, cat. no. M3148) !CAUTION 2-mercaptoethanol is toxic and should only be used in a fume hood.

    Ethanol Precipitation:

    Article Title: Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish
    Article Snippet: .. Purified mRNA was fragmented for 4 min at 94 °C using the NEBNext Magnesium RNA Fragmentation Module (NEB – E6150S) to approximately 200–500 bases, recovered by ethanol precipitation, and dissolved in 50 μl RNase-free water. .. TU-tagged RNA was biotinylated using EZ-Link HPDP-Biotin (Thermo Scientific – 21341) by the addition of 25 μl 4x TE and 25 μl 1 mg/ml EZ-Link (dissolved in DMF) to the 50 μl RNA.

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol. .. Standard ethanol precipitation was performed to precipitate the fragmented RNA.

    Spectrophotometry:

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: NanoDrop spectrophotometer (Thermo Scientific) was used to measure the concentration of RNA, and the integrity were tested by Agilent 2100 bioanalyzer. .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Concentration Assay:

    Article Title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
    Article Snippet: For Tie2:Cre; CA > GFPstop > UPRT experiments, 50 μg of total RNA was purified as described above, fragmented using NEBNext Magnesium RNA Fragmentation Module (New England BioLabs, #E6150S) for 4 min at 94°C, purified by RNeasy minikit (Qiagen, #74104), eluted in 50 μL of RNase-free water, processed through Ribo-Zero Magnetic kit (Epicenter, #MRZH116) for ribosomal RNA removal, purified by RNeasy minikit, and eluted in 22 μL of RNase-free water. .. The RNA concentration was determined by Qubit, and a 2-μL sample was reserved as the total RNA control.

    Article Title: BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing
    Article Snippet: The RNA concentration was determined using the Qubit RNA HS Assay Kit (Invitrogen, #Q32852), and integrity was assessed using a Fragment Analyzer (Advanced Analytical). .. RNA fragmentation was done using the NEBNext Magnesium RNA Fragmentation Module (NEB, #E6150S) with incubation time at 94 °C for 1 or 2 min.

    Article Title: mRNA N6-methyladenosine methylation of postnatal liver development in pig
    Article Snippet: Then, mRNA concentration was measured by Qubit 2.0 (Invitrogen) and the integrity was tested by agarose gel electrophoresis and Agilent 2100 bioanalyzer. .. The mRNA was chemically fragmented approximately 150-nucleotide-long using NEBNext® Magnesium RNA Fragmentation Module (NEB #E6150S) according to the manufacturer's protocol.

    Hood:

    Article Title: Applying thiouracil (TU)-tagging for mouse transcriptome analysis
    Article Snippet: .. Isopropyl alcohol (Macron Fine Chemicals, cat. no. MK303202) Ethanol (EtOH) (Pharmco Aaper, cat. no. 11100020G) RNase-free water (H2 O) (Ambion cat. no. AM9938) 1M Tris pH 8.0 RNase-free (Ambion cat. no. AM9855G) 0.5M EDTA pH 8.0 RNase-free (Ambion cat. no. AM9260G) TURBO DNase (Invitrogen cat. no. AM2238) RNeasy Mini Kit (Qiagen cat. no. 74101) Qubit RNA BR Assay Kit (Invitrogen, cat. no. ) Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. ) Qubit assay tubes (Invitrogen, cat. no. ) NEBNext magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) Ribo-Zero Magnetic Kit (Epicentre, cat. no. MRZH11124) N,N-Dimethylformamide (Sigma, cat. no. D4551) EZ-Link Biotin-HDPD (Thermo Scientific, cat. no. 21341) μMacs Streptavidin Kit (Miltenyi Biotec, cat. no. 130-074-101) 2-mercaptoethanol (Sigma, cat. no. M3148) !CAUTION 2-mercaptoethanol is toxic and should only be used in a fume hood. ..

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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: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    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