rrna depletion kit  (New England Biolabs)


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    Name:
    NEBNext rRNA Depletion Kit Bacteria
    Description:
    The NEBNext rRNA Depletion Kits Bacteria employ the NEBNext RNase H based RNA depletion workflow to deplete ribosomal rRNA 5S 16S and 23S from gram positive and gram negative organisms The kit is effective with both intact and degraded RNA preparations from monocultures or samples with mixed bacterial species
    Catalog Number:
    e7850l
    Price:
    1080
    Size:
    24 rxns
    Category:
    RNA Purification Kit Components
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    New England Biolabs rrna depletion kit
    NEBNext rRNA Depletion Kit Bacteria
    The NEBNext rRNA Depletion Kits Bacteria employ the NEBNext RNase H based RNA depletion workflow to deplete ribosomal rRNA 5S 16S and 23S from gram positive and gram negative organisms The kit is effective with both intact and degraded RNA preparations from monocultures or samples with mixed bacterial species
    https://www.bioz.com/result/rrna depletion kit/product/New England Biolabs
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    rrna depletion kit - by Bioz Stars, 2020-04
    99/100 stars

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    1) Product Images from "m6A enhances the phase separation potential of mRNA"

    Article Title: m6A enhances the phase separation potential of mRNA

    Journal: Nature

    doi: 10.1038/s41586-019-1374-1

    Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.
    Figure Legend Snippet: Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.

    Techniques Used: Knock-Out, Thin Layer Chromatography, Derivative Assay

    Related Articles

    Functional Assay:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: However, because more than 80% of the total RNA is rRNA, efficient functional profiling of rumen microbiome requires removal of the rRNA from the total RNA preparation. .. Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA.

    Amplification:

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. .. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).

    RNA Sequencing Assay:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: RNA-Seq is the preferred technology to perform metatranscriptomic analysis of rumen microbiome. .. Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA.

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics
    Article Snippet: Paragraph title: RNA-seq ... The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. .. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).

    Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease
    Article Snippet: RNA preparation and sequencing For the RNA sequencing analysis, total RNA was extracted from colon of mice using ISOGEN II reagent (Nippon Gene) according to the manufacturer’s instruction. .. In all, 500 ng of total RNA were ribosomal RNA-depleted using NEBNext rRNA Depletion Kit (New England Biolabs), and converted to Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs).

    Article Title: m6A enhances the phase separation potential of mRNA
    Article Snippet: Paragraph title: RNA-seq analysis ... Library preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit starting from 1 μg of total input RNA and following the protocol for use with NEBNext rRNA Depletion Kit.

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo
    Article Snippet: Paragraph title: RNA-seq ... Input of iCLIP samples were sequences similarly except that instead of polyA RNA enrichment, rRNAs were depleted using the NEBNext rRNA depletion kit (NEB).

    Isolation:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: .. Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA. .. Because of the difficulty in isolating high-quality RNA and depletion of rRNA, metatranscriptomics has just been used in a few studies, including the study that characterized the functional profiles of the consortia adherent to the bovine rumen [ , ].

    Article Title: The TAB1-p38 α complex aggravates myocardial injury and can be targeted by small molecules
    Article Snippet: RNA isolated from the 10 left ventricles (5 for each condition: WT vs. KI) was quantified using Qubit fluorometric quantification and assessed for quality using Agilent Bioanalyzer. .. Library preparation was performed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina and the NEBNext rRNA Depletion kit (New England BioLabs).

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics
    Article Snippet: Polyadenylated (PolyA+) RNA was captured with the NEBNext Poly(A) mRNA Magnetic Isolation Module. .. The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo
    Article Snippet: RNA-seq PolyA RNAs were isolated using NEBNext ploy(A) mRNA magnetic isolation module (NEB), and the libraries were generated using NEBNext Ultra directional RNA library prep kit (NEB). .. Input of iCLIP samples were sequences similarly except that instead of polyA RNA enrichment, rRNAs were depleted using the NEBNext rRNA depletion kit (NEB).

    Next-Generation Sequencing:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: Paragraph title: NEXT-GENERATION SEQUENCING ... Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA.

    Article Title: The TAB1-p38 α complex aggravates myocardial injury and can be targeted by small molecules
    Article Snippet: Paragraph title: Library preparation and next-generation sequencing. ... Library preparation was performed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina and the NEBNext rRNA Depletion kit (New England BioLabs).

    Sequencing:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: Therefore, metatranscriptomics is better suited than shotgun metagenomic sequencing in functional analysis of rumen microbiome. .. Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA.

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics
    Article Snippet: The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit). .. PolyA+ and PolyA− RNA libraries underwent sequencing (50 bp paired-end directional reads; ∼25 M reads/sample) on an Illumina sequencing platform (HiSeq2000).

    Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease
    Article Snippet: .. In all, 500 ng of total RNA were ribosomal RNA-depleted using NEBNext rRNA Depletion Kit (New England Biolabs), and converted to Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). .. The library was validated with Bioanalyzer (Agilent Technologies) to determine size distribution and concentration and sequenced on the NextSeq 500 (Illumina) with paired-end 36-base read option.

    Flow Cytometry:

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo
    Article Snippet: RNA-seqs were performed on an Illumina hiseq 2000 flow cell with a 150 nt run length. .. Input of iCLIP samples were sequences similarly except that instead of polyA RNA enrichment, rRNAs were depleted using the NEBNext rRNA depletion kit (NEB).

    Quantitative RT-PCR:

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: RNA was then analyzed either by RT-qPCR to measure α-satellite RNA levels as described above, or a cDNA library was generated for RNA-Seq analysis. .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions.

    Purification:

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics
    Article Snippet: RNA was extracted, DNase-treated and purified (RNeasy, Qiagen). .. The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. .. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).

    Real-time Polymerase Chain Reaction:

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. .. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).

    Concentration Assay:

    Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease
    Article Snippet: In all, 500 ng of total RNA were ribosomal RNA-depleted using NEBNext rRNA Depletion Kit (New England Biolabs), and converted to Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). .. The library was validated with Bioanalyzer (Agilent Technologies) to determine size distribution and concentration and sequenced on the NextSeq 500 (Illumina) with paired-end 36-base read option.

    Generated:

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics
    Article Snippet: Two distinct RNA libraries were generated. .. The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. .. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo
    Article Snippet: RNA-seq PolyA RNAs were isolated using NEBNext ploy(A) mRNA magnetic isolation module (NEB), and the libraries were generated using NEBNext Ultra directional RNA library prep kit (NEB). .. Input of iCLIP samples were sequences similarly except that instead of polyA RNA enrichment, rRNAs were depleted using the NEBNext rRNA depletion kit (NEB).

    RNA Expression:

    Article Title: m6A enhances the phase separation potential of mRNA
    Article Snippet: In order to measure RNA expression after stress, heat-shocked cells were placed back at 37°C for 1 h. After collecting cells as described in the Ribo-seq section, 5% of the lysate cells before RNase digestion was used to extract Total RNA. .. Library preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit starting from 1 μg of total input RNA and following the protocol for use with NEBNext rRNA Depletion Kit.

    Expressing:

    Article Title: Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction
    Article Snippet: HEAT MAP: Red indicate higher level of absolute expression. .. Kit abbreviations: RZ = RiboZero Gold, LX = Lexogen RiboCop, NE = NEBNext rRNA Depletion, K=Kapa RiboErase, CR = Clontech Ribogone, CZ = SMARTer Pico total RNA.

    Article Title: ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo
    Article Snippet: Reads were trimmed with fastx-toolkit, and mapped against human genome (hg19) with Tophat2, and gene expression was quantified by RPM using HTseq ( ). .. Input of iCLIP samples were sequences similarly except that instead of polyA RNA enrichment, rRNAs were depleted using the NEBNext rRNA depletion kit (NEB).

    cDNA Library Assay:

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin
    Article Snippet: RNA was then analyzed either by RT-qPCR to measure α-satellite RNA levels as described above, or a cDNA library was generated for RNA-Seq analysis. .. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions.

    Shotgun Sequencing:

    Article Title: Metagenomic investigation of gastrointestinal microbiome in cattle
    Article Snippet: One limitation of the shotgun sequencing approach is that it can only reveal the functional potential, not the actual functions expressed in any microbiome. .. Several commercial kits, such as the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, USA), MICROBExpress Bacterial mRNA Enrichment Kit (Ambion, Austin, TX, USA), RiboMinus Transcriptome Isolation Kit (Life Technologies, Gaithersburg, MD, USA), and NEBNext rRNA Depletion Kit (New England Biolabs, Beverly, MA, USA), are available to achieve a reasonably adequate depletion of rRNA.

    Mouse Assay:

    Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease
    Article Snippet: RNA preparation and sequencing For the RNA sequencing analysis, total RNA was extracted from colon of mice using ISOGEN II reagent (Nippon Gene) according to the manufacturer’s instruction. .. In all, 500 ng of total RNA were ribosomal RNA-depleted using NEBNext rRNA Depletion Kit (New England Biolabs), and converted to Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs).

    other:

    Article Title: Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction
    Article Snippet: These kits include NEBNext rRNA depletion, Kapa RiboErase, and Takara/Clontech’s RiboGone.

    Article Title: Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction
    Article Snippet: Dark Blue = RZ|RiboZero Gold, Yellow = LX|Lexogen RiboCop, Aqua = NE|NEBNext rRNA Depletion, Green = Q|Qiagen, Grey = K|Kapa RiboErase, Blue = CR|Clontech Ribogone, Orange = CZ|SMARTer Pico total RNA.

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    New England Biolabs rrna depletion kit
    Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A <t>RNA.</t> We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from <t>rRNA</t> or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.
    Rrna Depletion Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rrna depletion kit/product/New England Biolabs
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    rrna depletion kit - by Bioz Stars, 2020-04
    99/100 stars
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    The NEBNext Globin rRNA Depletion Kits Human Mouse Rat employ the NEBNext RNase H based RNA depletion workflow to deplete globin mRNA cytoplasmic rRNA and mitochondrial rRNA from human mouse
      Buy from Supplier

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    Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.

    Journal: Nature

    Article Title: m6A enhances the phase separation potential of mRNA

    doi: 10.1038/s41586-019-1374-1

    Figure Lengend Snippet: Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.

    Article Snippet: Library preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit starting from 1 μg of total input RNA and following the protocol for use with NEBNext rRNA Depletion Kit.

    Techniques: Knock-Out, Thin Layer Chromatography, Derivative Assay

    Regulation of mRNA and ecRNA by neuronal activity. ( a ) PolyA+ RNA-seq following 1 h neuronal depolarization (25 mM KCl) or inactivation (1 μM TTX) reveals altered mRNA expression at a small subset of genes. Top, heatmap of KCl-altered transcripts (each column=1 biological replicate; 2 replicates per treatment). Bottom, Venn diagram of overlap between transcripts altered by KCl and TTX. ( b ) Corresponding heatmaps from PolyA− RNA-seq reveal relationship between activity-related mRNA and ecRNA changes. PolyA− RNA transcription from 5′, intronic and 3′ sites all correlated significantly with mRNA changes following neuronal depolarization with KCl (linear regression, P

    Journal: Nature Communications

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics

    doi: 10.1038/ncomms12091

    Figure Lengend Snippet: Regulation of mRNA and ecRNA by neuronal activity. ( a ) PolyA+ RNA-seq following 1 h neuronal depolarization (25 mM KCl) or inactivation (1 μM TTX) reveals altered mRNA expression at a small subset of genes. Top, heatmap of KCl-altered transcripts (each column=1 biological replicate; 2 replicates per treatment). Bottom, Venn diagram of overlap between transcripts altered by KCl and TTX. ( b ) Corresponding heatmaps from PolyA− RNA-seq reveal relationship between activity-related mRNA and ecRNA changes. PolyA− RNA transcription from 5′, intronic and 3′ sites all correlated significantly with mRNA changes following neuronal depolarization with KCl (linear regression, P

    Article Snippet: The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Techniques: Activity Assay, RNA Sequencing Assay, Expressing

    Genome-wide identification and quantification of ecRNAs from neuronal systems. ( a ) RNA-seq workflow identifies both polyadenylated and non-polyadenylated transcripts from the same neuronal tissue. ( b ) Comparison of PolyA+ and PolyA− sequencing from representative gene loci reveals PolyA− transcripts arising from intronic and post-TESs. ( c ) Genome wide, extra-coding transcripts were characterized by averaging PolyA− reads that mapped to 5′ (pre-TSS), intronic or 3′ (post-TES) of a given gene. ( d ) Rank plot of ecRNA index at 17,719 rat genes. ( e ) mRNA expression (PolyA+ RNA-seq) ranked by ecRNA index reveals correlation between ecRNA and mRNA expression. ( f ) Division of ecRNA into discrete quartiles reveals general profile and expression of PolyA− RNA transcripts. Data are aligned to transcription start sites (TSS) and TESs. Heatmap shows PolyA− transcription from all genes. ( g ) MBD-seq reveals metagenomic DNA methylation profiles, including hypomethylation at TSS and hypermethylation at TES. ecRNA transcription is associated with hypomethylated promoters across the genome. ( h , i ) Genome wide, ecRNA levels are positively correlated with mRNA transcription (( h ) one-way ANOVA, F (3,17715) =612.5, P

    Journal: Nature Communications

    Article Title: Extra-coding RNAs regulate neuronal DNA methylation dynamics

    doi: 10.1038/ncomms12091

    Figure Lengend Snippet: Genome-wide identification and quantification of ecRNAs from neuronal systems. ( a ) RNA-seq workflow identifies both polyadenylated and non-polyadenylated transcripts from the same neuronal tissue. ( b ) Comparison of PolyA+ and PolyA− sequencing from representative gene loci reveals PolyA− transcripts arising from intronic and post-TESs. ( c ) Genome wide, extra-coding transcripts were characterized by averaging PolyA− reads that mapped to 5′ (pre-TSS), intronic or 3′ (post-TES) of a given gene. ( d ) Rank plot of ecRNA index at 17,719 rat genes. ( e ) mRNA expression (PolyA+ RNA-seq) ranked by ecRNA index reveals correlation between ecRNA and mRNA expression. ( f ) Division of ecRNA into discrete quartiles reveals general profile and expression of PolyA− RNA transcripts. Data are aligned to transcription start sites (TSS) and TESs. Heatmap shows PolyA− transcription from all genes. ( g ) MBD-seq reveals metagenomic DNA methylation profiles, including hypomethylation at TSS and hypermethylation at TES. ecRNA transcription is associated with hypomethylated promoters across the genome. ( h , i ) Genome wide, ecRNA levels are positively correlated with mRNA transcription (( h ) one-way ANOVA, F (3,17715) =612.5, P

    Article Snippet: The remaining non-polyadenylated (PolyA−) underwent ribosomal RNA depletion (NEBNext rRNA depletion kit).

    Techniques: Genome Wide, RNA Sequencing Assay, Sequencing, Expressing, DNA Methylation Assay