e7490  (New England Biolabs)


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    Name:
    NEBNext Poly A mRNA Magnetic Isolation Module
    Description:
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    Catalog Number:
    e7490l
    Price:
    242
    Size:
    96 rxns
    Category:
    mRNA Purification Kits
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    Structured Review

    New England Biolabs e7490
    NEBNext Poly A mRNA Magnetic Isolation Module
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    https://www.bioz.com/result/e7490/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e7490 - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Amplification:

    Article Title: Spatially resolved, highly multiplexed RNA profiling in single cells
    Article Snippet: .. Total RNA was extracted from IMR90 cells cultured as above using the Zymo Quick RNA MiniPrep kit (R1054) according to the manufacturer’s instructions. polyA RNA was then selected (NEB; E7490), and a sequencing library was constructed using the NEBNext Ultra RNA library preparation kit (NEB; E7530), amplified with custom oligonucleotides, and 150-bp reads were obtained from on a MiSeq. ..

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After Not I digestion, the library DNA was purified for PCR amplification (16 cycles) (more details in “Step-by-step Holo-Seq protocols” (Additional file )).

    Synthesized:

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. Briefly, the first-strand and the second of cDNA were synthesized using M-MuLV reverse transcriptase and DNA polymerase I (Invitrogen), respectively.

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: .. Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen). .. Quantitative reverse transcription-PCR (qRT-PCR) was performed using an ABI Prism 7900HT machine (Applied Biosystems, Foster City, CA).

    Construct:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). .. Illumina-compatible RNA-libraries were constructed by using the NEBNext Ultra RNALibrary Prep Kit for Illumina (E7530) as well as the NEBNext Multiplex Oligos for Illumina (E7335) following the distributors instruction manuals.

    Article Title: Spatially resolved, highly multiplexed RNA profiling in single cells
    Article Snippet: .. Total RNA was extracted from IMR90 cells cultured as above using the Zymo Quick RNA MiniPrep kit (R1054) according to the manufacturer’s instructions. polyA RNA was then selected (NEB; E7490), and a sequencing library was constructed using the NEBNext Ultra RNA library preparation kit (NEB; E7530), amplified with custom oligonucleotides, and 150-bp reads were obtained from on a MiSeq. ..

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: Three libraries labeled A, M and B were constructed from RNA extracted from the apical, middle and basal stem segments, respectively. .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490, E7530, E7420, and E7300) for the mRNA library, the directional total RNA library, and the small RNA library, respectively. .. Sequencing library construction of mRNAs from a single cell A single cell was lysed at 95 °C for 5 min in 0.1% BSA/PBS with 100 ng of the carrier RNA mixture.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After adaptor ligation and USER digestion, cDNA fragments from the RNA carrier were removed by Not I digestion.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The libraries were constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530), before PCR enrichment, the DNA was digested by Not I at 37 °C for 2 h or Cas9 nuclease with sgRNA mix at 37 °C for 2 h. .. In vitro digestion of the DNA template of carrier RNA using CRISPR/Cas9 The sgRNA sites on the DNA template were selected using the Optimized CRISPR Design website.

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: .. Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. Briefly, the first-strand and the second of cDNA were synthesized using M-MuLV reverse transcriptase and DNA polymerase I (Invitrogen), respectively.

    Real-time Polymerase Chain Reaction:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). .. Multiplexed RNA libraries were mixed in equilibrium according to their Qubit (Life Technologies) measurements, fragment distribution profiles on the Advanced Anayltical Fragment Analyzer and quantification of library 5′-and 3′- ends by Real Time PCR.

    Multiplex Assay:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). .. Illumina-compatible RNA-libraries were constructed by using the NEBNext Ultra RNALibrary Prep Kit for Illumina (E7530) as well as the NEBNext Multiplex Oligos for Illumina (E7335) following the distributors instruction manuals.

    Expressing:

    Article Title: A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation
    Article Snippet: From 2μg of total RNA from each biological replicate (4 MRTF-A−/− and 4 MRTF-A+/+ , age matched), Library preparation was done using a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) and mRNA enrichment was done by NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490), according to manufacturer's protocols. .. Fastq files are deposited at NCBI's Gene Expression Omnibus (GEO) database with BioProject ID PRJNA535475 and BioSample accession number SAMN11506732 .

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen). .. Results were normalized to hypoxanthine phosphoribosyltransferase expression and wild-type, untreated control animals.

    Flow Cytometry:

    Article Title: A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation
    Article Snippet: mRNA Sequencing The sequencing was performed at the Biomedicum Functional Genomics Unit (FuGU) of the University of Helsinki with the Illumina NextSeq500 using a NextSeq Mid Output 150 cycle flow cell. .. From 2μg of total RNA from each biological replicate (4 MRTF-A−/− and 4 MRTF-A+/+ , age matched), Library preparation was done using a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) and mRNA enrichment was done by NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490), according to manufacturer's protocols.

    Ligation:

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After adaptor ligation and USER digestion, cDNA fragments from the RNA carrier were removed by Not I digestion.

    Article Title: Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi
    Article Snippet: The NEBNext poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA; # E7490) was used to isolate intact poly(A)+ RNA from each previously isolated total RNA preparations. .. End repair and dA tailing of cDNA library was performed, immediately followed by adaptor ligation.

    Biomarker Assay:

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: Construction of the six libraries and RNA-Seq analysis were performed by Biomarker Biotechnology Corporation (Beijing, China). .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Cell Culture:

    Article Title: Spatially resolved, highly multiplexed RNA profiling in single cells
    Article Snippet: .. Total RNA was extracted from IMR90 cells cultured as above using the Zymo Quick RNA MiniPrep kit (R1054) according to the manufacturer’s instructions. polyA RNA was then selected (NEB; E7490), and a sequencing library was constructed using the NEBNext Ultra RNA library preparation kit (NEB; E7530), amplified with custom oligonucleotides, and 150-bp reads were obtained from on a MiSeq. ..

    Polymerase Chain Reaction:

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After Not I digestion, the library DNA was purified for PCR amplification (16 cycles) (more details in “Step-by-step Holo-Seq protocols” (Additional file )).

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The libraries were constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530), before PCR enrichment, the DNA was digested by Not I at 37 °C for 2 h or Cas9 nuclease with sgRNA mix at 37 °C for 2 h. .. In vitro digestion of the DNA template of carrier RNA using CRISPR/Cas9 The sgRNA sites on the DNA template were selected using the Optimized CRISPR Design website.

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. Next, these fragments were enriched by PCR amplifications and library quality was assessed by the Agilent Bioanalyzer 2100 System.

    Article Title: Lnc-mg is a long non-coding RNA that promotes myogenesis
    Article Snippet: Then polyA+ RNA fraction and polyA- RNA fraction were isolated by using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, NEB E7490S/L). .. In addition, the amount of lnc-mg was examined in PCR assay with polyA+ RNA fraction and polyA-RNA fraction, respectively.

    Immunofluorescence:

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: Paragraph title: Quantitative RT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry. ... Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen).

    RNA Sequencing Assay:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Paragraph title: High-throughput sequencing of RNA (RNA-Seq) ... Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490).

    Article Title: Spatially resolved, highly multiplexed RNA profiling in single cells
    Article Snippet: Paragraph title: Bulk RNA sequencing ... Total RNA was extracted from IMR90 cells cultured as above using the Zymo Quick RNA MiniPrep kit (R1054) according to the manufacturer’s instructions. polyA RNA was then selected (NEB; E7490), and a sequencing library was constructed using the NEBNext Ultra RNA library preparation kit (NEB; E7530), amplified with custom oligonucleotides, and 150-bp reads were obtained from on a MiSeq.

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: Paragraph title: RNA extraction, library construction and RNA-Seq ... The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5
    Article Snippet: Paragraph title: RNA-seq library preparation and sequencing ... After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: Paragraph title: Not I and Cas9 digestion of bulk RNA-Seq libraries ... The libraries were constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530), before PCR enrichment, the DNA was digested by Not I at 37 °C for 2 h or Cas9 nuclease with sgRNA mix at 37 °C for 2 h.

    Isolation:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: .. Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). .. Illumina-compatible RNA-libraries were constructed by using the NEBNext Ultra RNALibrary Prep Kit for Illumina (E7530) as well as the NEBNext Multiplex Oligos for Illumina (E7335) following the distributors instruction manuals.

    Article Title: A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation
    Article Snippet: .. From 2μg of total RNA from each biological replicate (4 MRTF-A−/− and 4 MRTF-A+/+ , age matched), Library preparation was done using a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) and mRNA enrichment was done by NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490), according to manufacturer's protocols. ..

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: RNA extraction, library construction and RNA-Seq Total RNA from each sample was isolated using CTAB plus the OMEGA Plant RNA isolation kit as described previously [ ]. .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5
    Article Snippet: .. After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction. .. The samples were sequenced on an Illumina HiSeq 2500 with 100-bp paired-end reads at the Vincent J. Coates Genomics Sequencing Laboratory at the University of California at Berkeley.

    Article Title: Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi
    Article Snippet: .. The NEBNext poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA; # E7490) was used to isolate intact poly(A)+ RNA from each previously isolated total RNA preparations. .. The eluted RNA was used for first and second strand cDNA synthesis using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA; # E7530).

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: .. Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. Briefly, the first-strand and the second of cDNA were synthesized using M-MuLV reverse transcriptase and DNA polymerase I (Invitrogen), respectively.

    Article Title: Lnc-mg is a long non-coding RNA that promotes myogenesis
    Article Snippet: .. Then polyA+ RNA fraction and polyA- RNA fraction were isolated by using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, NEB E7490S/L). .. In addition, the amount of lnc-mg was examined in PCR assay with polyA+ RNA fraction and polyA-RNA fraction, respectively.

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: .. Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen). .. Quantitative reverse transcription-PCR (qRT-PCR) was performed using an ABI Prism 7900HT machine (Applied Biosystems, Foster City, CA).

    Immunohistochemistry:

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: Paragraph title: Quantitative RT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry. ... Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen).

    Labeling:

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: Three libraries labeled A, M and B were constructed from RNA extracted from the apical, middle and basal stem segments, respectively. .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Purification:

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After Not I digestion, the library DNA was purified for PCR amplification (16 cycles) (more details in “Step-by-step Holo-Seq protocols” (Additional file )).

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. After NEBNext adaptors were ligated to the adenylated 3′ ends of cDNA, fragments of approximately 500 bp in length were purified and selected with the AMPure XP system (Beckman Coulter, Beverly, MA, United States).

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: .. Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen). .. Quantitative reverse transcription-PCR (qRT-PCR) was performed using an ABI Prism 7900HT machine (Applied Biosystems, Foster City, CA).

    Sequencing:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Paragraph title: High-throughput sequencing of RNA (RNA-Seq) ... Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490).

    Article Title: A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation
    Article Snippet: Paragraph title: mRNA Sequencing ... From 2μg of total RNA from each biological replicate (4 MRTF-A−/− and 4 MRTF-A+/+ , age matched), Library preparation was done using a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) and mRNA enrichment was done by NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490), according to manufacturer's protocols.

    Article Title: Spatially resolved, highly multiplexed RNA profiling in single cells
    Article Snippet: .. Total RNA was extracted from IMR90 cells cultured as above using the Zymo Quick RNA MiniPrep kit (R1054) according to the manufacturer’s instructions. polyA RNA was then selected (NEB; E7490), and a sequencing library was constructed using the NEBNext Ultra RNA library preparation kit (NEB; E7530), amplified with custom oligonucleotides, and 150-bp reads were obtained from on a MiSeq. ..

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490, E7530, E7420, and E7300) for the mRNA library, the directional total RNA library, and the small RNA library, respectively. .. Sequencing library construction of mRNAs from a single cell A single cell was lysed at 95 °C for 5 min in 0.1% BSA/PBS with 100 ng of the carrier RNA mixture.

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5
    Article Snippet: .. After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction. .. The samples were sequenced on an Illumina HiSeq 2500 with 100-bp paired-end reads at the Vincent J. Coates Genomics Sequencing Laboratory at the University of California at Berkeley.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The sequencing library was constructed following the manufacturer’s protocol using NEBNext kits (E7490 & E7530). .. After adaptor ligation and USER digestion, cDNA fragments from the RNA carrier were removed by Not I digestion.

    Article Title: Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation
    Article Snippet: .. Libraries Construction and Sequencing Before mRNA sequencing, poly (A) mRNA was isolated from total RNA using the NEB-E7490 Poly (A) mRNA Magnetic Isolation Kit (NEB, United States), and then fragmented by the Fragmentation Kit (Ambion, Austin, TX, United States). cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, United States) according to the manufacturer’s recommendations. .. Briefly, the first-strand and the second of cDNA were synthesized using M-MuLV reverse transcriptase and DNA polymerase I (Invitrogen), respectively.

    Quantitative RT-PCR:

    Article Title: Chromatin Profiling Reveals Regulatory Network Shifts and a Protective Role for Hepatocyte Nuclear Factor 4α during Colitis
    Article Snippet: Paragraph title: Quantitative RT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry. ... Five micrograms of total RNA from control and DSS-inflamed colon was used to purify poly(A) mRNA with the NEBNext poly(A) mRNA magnetic isolation module (E7490S/L; New England Biolabs). cDNA was synthesized from purified Poly(A) mRNA by using oligo(dT) primers (SuperScript III first-strand synthesis kit; Invitrogen).

    cDNA Library Assay:

    Article Title: Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi
    Article Snippet: The NEBNext poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA; # E7490) was used to isolate intact poly(A)+ RNA from each previously isolated total RNA preparations. .. End repair and dA tailing of cDNA library was performed, immediately followed by adaptor ligation.

    RNA Extraction:

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: Paragraph title: RNA extraction, library construction and RNA-Seq ... The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Functional Assay:

    Article Title: A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation
    Article Snippet: mRNA Sequencing The sequencing was performed at the Biomedicum Functional Genomics Unit (FuGU) of the University of Helsinki with the Illumina NextSeq500 using a NextSeq Mid Output 150 cycle flow cell. .. From 2μg of total RNA from each biological replicate (4 MRTF-A−/− and 4 MRTF-A+/+ , age matched), Library preparation was done using a NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760) and mRNA enrichment was done by NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490), according to manufacturer's protocols.

    Agarose Gel Electrophoresis:

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: The RNA samples were checked for integrity on a 1.2% agarose gel and quantified using a Nanodrop 1000 spectrophotometer. .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    Article Title: Lnc-mg is a long non-coding RNA that promotes myogenesis
    Article Snippet: Next, the RNA quality was assessed by formaldehyde agarose gel electrophoresis, quantified spectrophotometrically and Agilent 2200 Bioanalyzer (Agilent). .. Then polyA+ RNA fraction and polyA- RNA fraction were isolated by using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, NEB E7490S/L).

    Spectrophotometry:

    Article Title: Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq
    Article Snippet: The RNA samples were checked for integrity on a 1.2% agarose gel and quantified using a Nanodrop 1000 spectrophotometer. .. The mRNA enrichment and library construction were carried out according to protocol of NEB kit (E7490, E6110, E7500).

    High Throughput Screening Assay:

    Article Title: Queuine links translational control in eukaryotes to a micronutrient from bacteria
    Article Snippet: Paragraph title: High-throughput sequencing of RNA (RNA-Seq) ... Inc; Ankeny Iowa, USA), the mRNA was isolated from high quality total RNA extracts by using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490).

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  • 95
    New England Biolabs mrna magnetic isolation module
    The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing <t>RNA</t> binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC <t>mRNA</t> levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P
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    The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, RNA Binding Assay, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test

    IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Expressing, Binding Assay, Methylation, Real-time Polymerase Chain Reaction, Modification, Luciferase, Activity Assay, In Vivo, Plasmid Preparation, Two Tailed Test

    Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, Modification, Affinity Chromatography, Methylation, Silver Staining, Western Blot, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Cross-linking Immunoprecipitation, In Vivo

    This schematic illustrates the sample (auburn rectangle) and library (blue rectangle) preparation workflow to generate the libraries that were loaded on the Illumina sequencer. ( a ) For B. malayi and A. fumigatus , a poly(A)-selected sample was created from an aliquot of total RNA that was used to create a poly(A)-selected library. ( b ) The B. malayi or A. fumigatus AgSS baits were subsequently used to capture the targeted RNA from poly(A)-selected libraries. ( c ) For AgSS-enriched w Bm libraries, an RNA library was constructed from an aliquot of total RNA that underwent targeted enrichment with the Wolbachia AgSS baits. Unlike the eukaryotic enrichments, the bacterial AgSS capture is performed on total RNA. For a limited number of libraries described in the text, an RNA library was constructed from an aliquot of total RNA (i.e. without poly(A)-enrichment) that underwent targeted enrichment with the Brugia AgSS baits. ( d ) For poly(A)/rRNA-depleted libraries enriched for w Bm, an aliquot of total RNA from either mosquito thoraces or adult nematodes was enriched for bacterial mRNA by removing Gram-negative and human rRNAs with two RiboZero removal kits and polyadenylated RNAs with DynaBeads.

    Journal: Scientific Reports

    Article Title: Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses

    doi: 10.1038/s41598-018-31420-7

    Figure Lengend Snippet: This schematic illustrates the sample (auburn rectangle) and library (blue rectangle) preparation workflow to generate the libraries that were loaded on the Illumina sequencer. ( a ) For B. malayi and A. fumigatus , a poly(A)-selected sample was created from an aliquot of total RNA that was used to create a poly(A)-selected library. ( b ) The B. malayi or A. fumigatus AgSS baits were subsequently used to capture the targeted RNA from poly(A)-selected libraries. ( c ) For AgSS-enriched w Bm libraries, an RNA library was constructed from an aliquot of total RNA that underwent targeted enrichment with the Wolbachia AgSS baits. Unlike the eukaryotic enrichments, the bacterial AgSS capture is performed on total RNA. For a limited number of libraries described in the text, an RNA library was constructed from an aliquot of total RNA (i.e. without poly(A)-enrichment) that underwent targeted enrichment with the Brugia AgSS baits. ( d ) For poly(A)/rRNA-depleted libraries enriched for w Bm, an aliquot of total RNA from either mosquito thoraces or adult nematodes was enriched for bacterial mRNA by removing Gram-negative and human rRNAs with two RiboZero removal kits and polyadenylated RNAs with DynaBeads.

    Article Snippet: When targeting eukaryotic mRNA, polyadenylated RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module.

    Techniques: Construct

    Alternative pre-mRNA splicing of RPL7A mRNA is regulated by hnRNPA1 and DDX5. ( A ) Genome browser shot of the RPL7A gene region with RNA-seq data upon hnRNPA1 knockdown ( top ) and DDX5 knockdown ( bottom ), with corresponding nuclear CLIP peak cluster regions shown below . ( B ) icSHAPE-constrained in vivo and in vitro secondary structures for the RPL7A RNA. The nuclear eCLIP cluster/peak region is highlighted in green, the hnRNPA1 cross-link sites are marked with red asterisks, and the DDX5 cross-link sites are marked with blue plus signs. The dotted red rectangles indicate regions of the RNA with secondary structural changes between the in vivo and in vitro icSHAPE constraints. The enriched hnRNPA1 motifs are indicated by orange lines, and the enriched GC-rich motifs for DDX5 is indicated by dark-green lines.

    Journal: Genes & Development

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5

    doi: 10.1101/gad.316034.118

    Figure Lengend Snippet: Alternative pre-mRNA splicing of RPL7A mRNA is regulated by hnRNPA1 and DDX5. ( A ) Genome browser shot of the RPL7A gene region with RNA-seq data upon hnRNPA1 knockdown ( top ) and DDX5 knockdown ( bottom ), with corresponding nuclear CLIP peak cluster regions shown below . ( B ) icSHAPE-constrained in vivo and in vitro secondary structures for the RPL7A RNA. The nuclear eCLIP cluster/peak region is highlighted in green, the hnRNPA1 cross-link sites are marked with red asterisks, and the DDX5 cross-link sites are marked with blue plus signs. The dotted red rectangles indicate regions of the RNA with secondary structural changes between the in vivo and in vitro icSHAPE constraints. The enriched hnRNPA1 motifs are indicated by orange lines, and the enriched GC-rich motifs for DDX5 is indicated by dark-green lines.

    Article Snippet: After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction.

    Techniques: RNA Sequencing Assay, Cross-linking Immunoprecipitation, In Vivo, In Vitro

    Quantitation of differential AS events controlled by hnRNPA1 from RNA-seq using JUM. ( A , left panel) siRNA-mediated knockdown of hnRNPA1 at the protein level. K562 cells were transfected with either nonspecific control siRNA oligos (scr si) or hnRNPA1 duplex siRNA oligos. After a second round of siRNA transfection, the cells were harvested for RNA isolation or protein lysates. ( Right panel) JUM is a splicing annotation-independent method for determining pre-mRNA splicing patterns from RNA-seq data. Only splice junction-spanning reads were taken into account for quantitation. This resulted in a quantitative comparison of AS events (1828) whose splicing patterns were significantly altered in the hnRNPA1 knockdown samples versus the control (false discovery rate [FDR], P

    Journal: Genes & Development

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5

    doi: 10.1101/gad.316034.118

    Figure Lengend Snippet: Quantitation of differential AS events controlled by hnRNPA1 from RNA-seq using JUM. ( A , left panel) siRNA-mediated knockdown of hnRNPA1 at the protein level. K562 cells were transfected with either nonspecific control siRNA oligos (scr si) or hnRNPA1 duplex siRNA oligos. After a second round of siRNA transfection, the cells were harvested for RNA isolation or protein lysates. ( Right panel) JUM is a splicing annotation-independent method for determining pre-mRNA splicing patterns from RNA-seq data. Only splice junction-spanning reads were taken into account for quantitation. This resulted in a quantitative comparison of AS events (1828) whose splicing patterns were significantly altered in the hnRNPA1 knockdown samples versus the control (false discovery rate [FDR], P

    Article Snippet: After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction.

    Techniques: Quantitation Assay, RNA Sequencing Assay, Transfection, Isolation

    The RNA helicase DDX controls alternative pre-mRNA splicing of thousands of target RNAs. ( A , left panel) K562 cells were transfected with either nonspecific control siRNA oligos (scr si) or DDX5 duplex siRNA oligos. After a second round of siRNA transfection, the cells were harvested for RNA isolation or protein lysates. Protein lysates were immunoblotted with DDX5 antibody to detect the efficiency of siRNA-mediated knockdown at the protein level. ( Right panel) Detection of changes in AS events upon DDX5 knockdown in human K562 cells using JUM. JUM analysis revealed 3915 AS events whose splicing patterns were significantly altered in DDX5 RNAi knockdown samples versus the control scrambled siRNA samples, covering 2804 genes (FDR, P

    Journal: Genes & Development

    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5

    doi: 10.1101/gad.316034.118

    Figure Lengend Snippet: The RNA helicase DDX controls alternative pre-mRNA splicing of thousands of target RNAs. ( A , left panel) K562 cells were transfected with either nonspecific control siRNA oligos (scr si) or DDX5 duplex siRNA oligos. After a second round of siRNA transfection, the cells were harvested for RNA isolation or protein lysates. Protein lysates were immunoblotted with DDX5 antibody to detect the efficiency of siRNA-mediated knockdown at the protein level. ( Right panel) Detection of changes in AS events upon DDX5 knockdown in human K562 cells using JUM. JUM analysis revealed 3915 AS events whose splicing patterns were significantly altered in DDX5 RNAi knockdown samples versus the control scrambled siRNA samples, covering 2804 genes (FDR, P

    Article Snippet: After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)+ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction.

    Techniques: Transfection, Isolation

    Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). .

    Journal: Cancer Cell

    Article Title: Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting

    doi: 10.1016/j.ccell.2018.08.017

    Figure Lengend Snippet: Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). .

    Article Snippet: For RNA-seq of C1R-CD1d parental cells, the same procedures were followed, except for the fact that libraries were prepared .using the NEBNext poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra RNA Library Prep kit for Illumina (New Engand Biolabs) and sequenced on an Illumina HiSeq 2500 platform to obtain paired-end 100bp reads.

    Techniques: Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation