nebnext multiplex oligos  (New England Biolabs)


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    Name:
    NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1
    Description:
    NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 96 rxns
    Catalog Number:
    e7600s
    Price:
    460
    Size:
    96 rxns
    Category:
    DNA Template Preparation for PCR
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    Structured Review

    New England Biolabs nebnext multiplex oligos
    NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1
    NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 96 rxns
    https://www.bioz.com/result/nebnext multiplex oligos/product/New England Biolabs
    Average 99 stars, based on 236 article reviews
    Price from $9.99 to $1999.99
    nebnext multiplex oligos - by Bioz Stars, 2020-04
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    Centrifugation:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing. .. The closed circular genome is 865,472 bp in length, with G+C content of 27.5 mol%, close to the 26.5 mol% estimated by using isopycnic centrifugation ( ).

    Amplification:

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: The thermocycler protocol was: 42 ˚C for 60 min then 94 ˚C for 2 min then 5 cycles of 94 ˚C for 30 s, 44 ˚C for 30 s, 68 ˚C for 3 min, then 28 cycles of 94 ˚C for 30 s, 57 ˚C for 30 s, 68 ˚C for 3 min. Amplification of all eight segments was confirmed by gel electrophoresis, and 750 ng of each cDNA mixture were sheared to an average size of 300 to 400 bp using a Covaris (Woburn, MA) S220 focused ultrasonicator. .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA).

    Article Title: Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
    Article Snippet: Next-generation sequencing (NGS) analysis of edited cells PCR amplicons were repaired, A-tailed, adapter ligated and amplified using the NEB (Ipswich, MA) Next Ultra kit (NEB E7370L). .. Dual indexing (NEB E7600S) was implemented to permit multiplex sequencing.

    Polymerase Chain Reaction:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: DNA was purified using Agencourt AMPure XP PCR purification beads (Beckman coulter), and the DNA products were sequentially cut with NsiI-HF and AFlIIII to produce a ∼435bp product. .. The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions.

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Article Title: Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
    Article Snippet: Next-generation sequencing (NGS) analysis of edited cells PCR amplicons were repaired, A-tailed, adapter ligated and amplified using the NEB (Ipswich, MA) Next Ultra kit (NEB E7370L). .. Dual indexing (NEB E7600S) was implemented to permit multiplex sequencing.

    Construct:

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: The library was constructed with average insert lengths of 500 bp according to the manufacturer’s instruction (The DNA Library Prep Reagent Set for Illumina, E6000S/L, NEB). .. Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: .. Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs). .. Illumina MiSeq 2 × 300-bp paired-end sequencing yielded approximately 5 × 107 Phred quality-filtered reads with a quality score greater than 30.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing. ..

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. SSLs were constructed for at least one individual from each population.

    Incubation:

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Mass Spectrometry:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions. .. The DNA was sequenced on an Illumina MiSeq using MiSeq Reagent Kit v2 (500-cycles) (Illumina, MS-102-2003) PANDAseq was used to assemble paired-end FASTQ reads resulting from sequence analysis ( ).

    RAST Test:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs). .. The final assembly with coverage depth of more than 800× was achieved in May 2018 by using a combination of Ray v2.3.1, Unicycler v0.4.3, and Consed v29 finishing software ( , ) and then annotated with the RAST server and the NCBI Prokaryotic Genome Annotation Pipeline ( , ), all with default settings.

    Ligation:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: .. The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions. .. The DNA was again purified, quantified using Qubit dsDNA HS Assay Kit (Invitrogen, ).

    Genomic Sequencing:

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: The overhanging genomic sequences resulting from fragmentation were converted into blunt ends using T4 DNA polymerase, the Klenow fragment and T4 polynucleotide kinase. .. Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments.

    Northern Blot:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: ANNOUNCEMENT Episodic seal die-offs documented along northern coasts of the Atlantic Ocean since 1979 have been attributed to morbillivirus or influenzavirus outbreaks involving pneumonia with systemic disease ( , ). .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing.

    Generated:

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. DNA was sequenced on an Illumina HiSeq 2500 Sequencer (Illumina), with 100-bp paired-end reads generated for all but six samples, which had 100-bp single-end reads (Massively Parallel Sequencing Shared Resource Facility, Oregon Health and Science University, Portland, Oregon, USA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: Illumina library preparation and sequencing We amplified cDNA corresponding to all eight genomic segments from 5 μl of viral RNA using the SuperScript III One-Step RT-PCR Platinum Taq HiFi Kit (Invitrogen 12574). .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA).

    DNA Extraction:

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: After DNA extraction, DNA concentration was quantified on a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and pooled by population in an equimolar fashion (20 samples per PL). .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA).

    Nucleic Acid Electrophoresis:

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: Genome Sequencing of 919TP from Phage Particles and of Its Host Strain V. cholerae 919T After the DNA sample was extracted, the sample quality was analyzed by gel electrophoresis. .. Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments.

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: The thermocycler protocol was: 42 ˚C for 60 min then 94 ˚C for 2 min then 5 cycles of 94 ˚C for 30 s, 44 ˚C for 30 s, 68 ˚C for 3 min, then 28 cycles of 94 ˚C for 30 s, 57 ˚C for 30 s, 68 ˚C for 3 min. Amplification of all eight segments was confirmed by gel electrophoresis, and 750 ng of each cDNA mixture were sheared to an average size of 300 to 400 bp using a Covaris (Woburn, MA) S220 focused ultrasonicator. .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA).

    Isolation:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: Mycoplasma phocidae strain 105T was first isolated from the lungs of one of more than 400 harbor seals ( Phoca vitulina ) that died along the U.S. New England coastline during a mass mortality event in 1980. .. Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs).

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: Mycoplasma phocirhinis was first isolated from the respiratory tracts and hearts from harbor seals ( Phoca vitulina L.) that died along the adjacent North and Baltic seacoasts during a mass mortality event in 1988 ( ). .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing.

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. Residual primer dimers were removed by gel isolation of a 300–500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. Residual primer dimers were removed by gel isolation of a 300 to 500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

    Purification:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: .. The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions. .. The DNA was again purified, quantified using Qubit dsDNA HS Assay Kit (Invitrogen, ).

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: The DNA sample was then used to construct a library following shearing of the purified DNA into smaller fragments of the desired size using a Covaris S/E210 focused-ultrasonicator. .. Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments.

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. Residual primer dimers were removed by gel isolation of a 300–500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. Residual primer dimers were removed by gel isolation of a 300 to 500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

    Sequencing:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: Paragraph title: Library preparation and analysis for Ighv NGS sequencing ... The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions.

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: Paragraph title: Genome Sequencing of 919TP from Phage Particles and of Its Host Strain V. cholerae 919T ... Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: .. Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs). .. Illumina MiSeq 2 × 300-bp paired-end sequencing yielded approximately 5 × 107 Phred quality-filtered reads with a quality score greater than 30.

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing. ..

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific, Waltham, MA), and equal nanomolar concentrations were pooled.

    Article Title: Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
    Article Snippet: .. Dual indexing (NEB E7600S) was implemented to permit multiplex sequencing. .. All samples were pooled in equimolar amounts and sequenced on an Illumina (San Diego, CA) MiSeq using the MiSeq Reagent Kit v3 (2 × 300).

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. DNA was sequenced on an Illumina HiSeq 2500 Sequencer (Illumina), with 100-bp paired-end reads generated for all but six samples, which had 100-bp single-end reads (Massively Parallel Sequencing Shared Resource Facility, Oregon Health and Science University, Portland, Oregon, USA).

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific), and equal nanomolar concentrations were pooled.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. ) .. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, cat.no. )

    Plasmid Preparation:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: E3110K) Plasmid-Safe 10× Reaction Buffer (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase 25mM ATP solution (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase Cas9 nuclease S. pyogenes (New England BioLabs, cat.no M0386M) 10× Cas9 buffer (New England BioLabs), supplied with Cas9 nuclease S. pyogenes NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England BioLabs, cat.no. .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. )

    Software:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs). .. The final assembly with coverage depth of more than 800× was achieved in May 2018 by using a combination of Ray v2.3.1, Unicycler v0.4.3, and Consed v29 finishing software ( , ) and then annotated with the RAST server and the NCBI Prokaryotic Genome Annotation Pipeline ( , ), all with default settings.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing. .. The final assembly, with a coverage depth of 250×, was achieved by using a combination of PEAR v0.9.11, Ray v2.3.1, Edena v3.131028, and Circlator v1.5.5 software ( ) and then annotated starting at the dnaA gene via the RASTtk and NCBI Prokaryotic Genome Annotation pipelines ( , ), all with default settings.

    Multiplex Assay:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: .. The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions. .. The DNA was again purified, quantified using Qubit dsDNA HS Assay Kit (Invitrogen, ).

    Article Title: The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family
    Article Snippet: .. Following addition of an ‘A’ base to the 3′ end of the blunt phosphorylated DNA fragments, adapters (The NEBNext Multiplex Oligos for Illumina, E7600S, NEB) were ligated to the ends of the DNA fragments. ..

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: .. Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs). .. Illumina MiSeq 2 × 300-bp paired-end sequencing yielded approximately 5 × 107 Phred quality-filtered reads with a quality score greater than 30.

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Article Title: Tracking transcriptomic responses to endogenous and exogenous variation in cetaceans in the Southern California Bight
    Article Snippet: .. Libraries were prepared for RNAseq analysis using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (#E7420S) and indexed with NEBNext Multiplex Oligos for Illumina (#E7600S, New England Biolabs, Ipswich, MA, USA). .. Library quality was checked on an Agilent Bioanalyzer and quantified via Qubit fluorometer.

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing. ..

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific, Waltham, MA), and equal nanomolar concentrations were pooled.

    Article Title: Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
    Article Snippet: .. Dual indexing (NEB E7600S) was implemented to permit multiplex sequencing. .. All samples were pooled in equimolar amounts and sequenced on an Illumina (San Diego, CA) MiSeq using the MiSeq Reagent Kit v3 (2 × 300).

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. SSLs were constructed for at least one individual from each population.

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: .. Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific), and equal nanomolar concentrations were pooled.

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. ) .. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, cat.no. )

    Selection:

    Article Title: An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
    Article Snippet: .. Sequencing libraries were prepared from 500 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog number E7490, protocol revision 5.0), NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (catalog number E7420, protocol revision 6.0), and NEBNext Multiplex Oligos for Illumina (catalog number E7600, protocol revision 2.0) with the following modifications: E7420, Section1.2 (“mRNA Isolation, Fragmentation and Priming Total RNA”) Step 37, we decreased the incubation time from 15 min to 5 min; E7420, Section 1.3 (“First Strand cDNA Synthesis”), Step 2,we increased the incubation time from 15 min to 50 min; for the size selection we used 40 µL AMPure XP beads for the first bead selection and 20 µL AMPure XP beads for the second bead selection (targeting an insert size of 300–450 bp and a final library size of 400–550 bp); and in E7420, Section 1.9A (“PCR library Enrichment”), Step 2 we used 14 cycles for PCR cycling and dual index primers (i507 and i705–i712). .. Sequencing was performed at UC Berkeley's QB3 Vincent J. Coates Genomics Sequencing Laboratory on an Illumina HiSeq4000 instrument, generating 150 bp paired end reads.

    Next-Generation Sequencing:

    Article Title: Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging
    Article Snippet: Paragraph title: Library preparation and analysis for Ighv NGS sequencing ... The DNA was subsequently purified, end prep and adaptor ligation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (New Enlgand Biolabs, E7645S) according to the manufacturer’s instructions and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, E7600S) were added according to the manufacturers instructions.

    Article Title: Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
    Article Snippet: Paragraph title: Next-generation sequencing (NGS) analysis of edited cells ... Dual indexing (NEB E7600S) was implemented to permit multiplex sequencing.

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: Paragraph title: Next-generation sequencing ... Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S).

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: Paragraph title: CIRCLE-seq library preparation and NGS ... E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. )

    Concentration Assay:

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific, Waltham, MA), and equal nanomolar concentrations were pooled.

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: After DNA extraction, DNA concentration was quantified on a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and pooled by population in an equimolar fashion (20 samples per PL). .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA).

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. The final concentration of each barcoded library was determined by Quanti PicoGreen dsDNA quantification (ThermoFisher Scientific), and equal nanomolar concentrations were pooled.

    DNA Purification:

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocidae Strain 105T Genome
    Article Snippet: The DNA was extracted using an Easy-DNA genomic DNA purification kit (catalog number K180001; Thermo Fisher Scientific). .. Sequencing libraries were constructed using the NEBNext UltraTMII DNA library prep kit (catalog number E7645S; New England Biolabs) and dual-index NEBNext Multiplex Oligos (catalog number E7600S; New England Biolabs).

    Article Title: Complete Sequence and Annotation of the Mycoplasma phocirhinis Strain 852T Genome
    Article Snippet: Briefly, the DNA was extracted directly from a lyophilized specimen of M. phocirhinis ( ATCC catalog no. 49639) using an Easy-DNA genomic DNA purification kit (Thermo Fisher Scientific catalog no. K180001). .. Libraries were constructed using the NEBNext UltraTMII DNA library prep kit (New England Biolabs catalog no. E7645S) and dual-index NEBNext multiplex oligos (New England Biolabs catalog no. E7600S) for Illumina MiSeq 2 × 150-bp paired-end sequencing.

    Gel Extraction:

    Article Title: Stochastic processes constrain the within and between host evolution of influenza virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter A63881, Indianapolis, IN), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S, Ipswich, MA). .. Residual primer dimers were removed by gel isolation of a 300–500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

    Article Title: A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus
    Article Snippet: Sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit (NEB E7370L), Agencourt AMPure XP beads (Beckman Coulter ), and NEBNext multiplex oligonucleotides for Illumina (NEB E7600S). .. Residual primer dimers were removed by gel isolation of a 300 to 500 bp band, which was purified using a GeneJet Gel Extraction Kit (ThermoFisher Scientific).

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    New England Biolabs ultra ii dna library prep kit
    Identification and global characterization of <t>DNA-associated</t> RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in <t>HeLa</t> S3 cells. Data are from HeLa S3 cells. Adjusted P -values
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ultra rna library prep kit
    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. <t>RNA</t> was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense <t>oligos</t> (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P
    Ultra Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 945 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultra ii directional rna library prep kit
    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear <t>RNA</t> (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA <t>oligos</t> covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.
    Ultra Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification and global characterization of DNA-associated RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in HeLa S3 cells. Data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Identification and global characterization of DNA-associated RNA. ( A ) Scatter plots showing the correlation between TriplexRNA isolated by DNA-IP and SPRI selection (left) and TriplexRNA (DNA-IP) and control RNA (middle, right). Pearson correlation coefficient ( R ) across 7148 genes overlapping peaks is shown. Green diagonal line x = y . Some representative genes that overlap TriplexRNAs and control RNAs are highlighted. ( B ) Pie charts depicting the genomic distribution of TriplexRNA (DNA-IP) compared to chromatin-associated and nuclear RNA peaks, excluding intronic and exonic gene regions. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. ( C ) Pie chart showing classification of long noncoding RNAs that overlap TriplexRNA (DNA-IP), chromatin-associated and nuclear RNA. ( D ) Association of TriplexRNA (DNA-IP) and control RNA with ChromHMM promoter states and transcribed states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong (Tx) and weak (TxWk) transcription regions are shown. ( E ) Left: Overlap of TriplexRNA (DNA-IP), chromatin-associated RNA and nuclear RNA with different classes of repeat elements. Right: Abundance of simple repeat subclasses. ( F ) Abundance of TriplexRNA (DNA-IP) and control RNAs overlapping super-enhancers in HeLa S3 cells. Data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Isolation, Selection

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Quantitative RT-PCR, Purification, Polyacrylamide Gel Electrophoresis, Labeling, Selection, Isolation, RNA Sequencing Assay

    Isolation and identification of RNA-associated DNAs. ( A ) Scheme illustrating the method used to isolate RNA-associated DNA (TriplexDNA). ( B ) Pie chart depicting the genomic distribution of TriplexDNA peaks. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. The bar diagrams at the right display the fold change in the distribution of the respective regions in TriplexDNA compared to control DNA. ( C ) Line plots depicting the mean values of TriplexDNA-seq signals over TSS and TTS of 890 genes that overlap RNA-associated DNA peaks. Interval defined by maximum and minimum values is shaded. ( D ) TriplexDNA-seq regions overlapping DNase Hypersensitive Sites (DNase HS) in HeLa S3 cells provided by ENCODE. ( E ) Abundance of TriplexDNA regions associated with the indicated ChromHMM states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong and weak (Tx, TxWk) transcription, heterochromatin (Het) and Polycomb-repressed (RepPC) regions are shown. ( F ) Top: Overlap of TriplexDNA and control DNA with different classes of repeat elements. Bottom: Abundance of predominating repeat subclasses in TriplexDNA. (G) MEME motif analysis identifying purine-rich consensus motifs in randomly selected 500 TriplexDNA peaks. Data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Isolation and identification of RNA-associated DNAs. ( A ) Scheme illustrating the method used to isolate RNA-associated DNA (TriplexDNA). ( B ) Pie chart depicting the genomic distribution of TriplexDNA peaks. Upstream and downstream regions are defined within 2.5 kb proximity of the closest gene. The bar diagrams at the right display the fold change in the distribution of the respective regions in TriplexDNA compared to control DNA. ( C ) Line plots depicting the mean values of TriplexDNA-seq signals over TSS and TTS of 890 genes that overlap RNA-associated DNA peaks. Interval defined by maximum and minimum values is shaded. ( D ) TriplexDNA-seq regions overlapping DNase Hypersensitive Sites (DNase HS) in HeLa S3 cells provided by ENCODE. ( E ) Abundance of TriplexDNA regions associated with the indicated ChromHMM states. Active transcription start site (TssA), flanking active TSS (TssAFlnk), strong and weak (Tx, TxWk) transcription, heterochromatin (Het) and Polycomb-repressed (RepPC) regions are shown. ( F ) Top: Overlap of TriplexDNA and control DNA with different classes of repeat elements. Bottom: Abundance of predominating repeat subclasses in TriplexDNA. (G) MEME motif analysis identifying purine-rich consensus motifs in randomly selected 500 TriplexDNA peaks. Data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: DNA libraries were prepared from at least three independent experiments from HeLa S3 cells using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Whisker Assay, Labeling

    Multiplex ligation-dependent probe amplification analysis of ectodermal dysplasia-associated genes. DNA was denatured and hybridized with SALSA probe mix, followed by ligation and polymerase chain reaction amplification. Capillary electrophoresis was performed to generate fragment length and peak area using GeneMapper version 3.0 software. Copy number ratio is denoted as the ratio of peak area of patient vs. peak area of references. Blue color peaks represent the patient sample, whereas the red color peaks were reference.

    Journal: Molecular Medicine Reports

    Article Title: Gene screening facilitates diagnosis of complicated symptoms: A case report

    doi: 10.3892/mmr.2017.7590

    Figure Lengend Snippet: Multiplex ligation-dependent probe amplification analysis of ectodermal dysplasia-associated genes. DNA was denatured and hybridized with SALSA probe mix, followed by ligation and polymerase chain reaction amplification. Capillary electrophoresis was performed to generate fragment length and peak area using GeneMapper version 3.0 software. Copy number ratio is denoted as the ratio of peak area of patient vs. peak area of references. Blue color peaks represent the patient sample, whereas the red color peaks were reference.

    Article Snippet: PCR was performed with a primer cocktail using NEBNext® Ultra™ II DNA Library Prep kit (New England BioLabs, Inc.), followed by purification.

    Techniques: Multiplex Assay, Ligation, Amplification, Polymerase Chain Reaction, Electrophoresis, Software

    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: In Vivo, Transfection, Metabolic Labelling, Agarose Gel Electrophoresis, In Vitro, Incubation, Nucleic Acid Electrophoresis, Cell Culture, Activity Assay, Purification, Mutagenesis, Infection, Expressing, shRNA, Allele-specific Oligonucleotide

    Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Journal: Nature Communications

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    doi: 10.1038/ncomms10734

    Figure Lengend Snippet: Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Article Snippet: The RNA-seq libraries were created using the NEBNext Ultra RNA Library Prep kit for Illumina (E7530) with NEBNext Multiplex Oligos for Illumina (E7300).

    Techniques: Northern Blot, Expressing, Activated Clotting Time Assay, Western Blot, Staining, Over Expression, Stable Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Cell Culture

    NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: NEAT1 forms triplexes at numerous genomic sites. ( A ) NEAT1 profiles in TriplexRNA-seq (DNA-IP) (red) and nuclear RNA (blue) from HeLa S3 and U2OS cells with shaded TFR1 and TFR2. Minus (-) and plus (+) strands are shown. The position and sequence of NEAT1-TFR1 and -TFR2 are shown below. ( B ) EMSAs using 10 or 100 pmol of synthetic NEAT1 versions comprising TFR1 (40 or 52 nt) or TFR2 incubated with 0.25 pmol of double–stranded 32 P-labeled oligonucleotides which harbor sequences of NEAT1 target genes predicted from CHART-seq ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control, RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( C ) Schematic depiction of the TFR-based capture assay. Biotinylated RNA oligos covering NEAT1-TFR1 and NEAT1-TFR2 were used to capture genomic DNA. ( D ) MEME motif analysis identifying consensus motifs in DNA captured by NEAT1-TFR1 (399 of top 500 peaks) and by NEAT1-TFR2 (500 of top 500 peaks ranked by peak P -value). Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). ( E ) TDF analysis of the triplex-forming potential of NEAT1-TFR1 and NEAT1-TFR2 RNAs with top 500 TFR-associated and control DNA peaks (ranked by peak P -value) compared to 500 randomized regions ( N = 1000, colored grey). P -values were obtained from one-tailed Mann–Whitney test. ( F ) Scheme presenting antisense oligo (ASO)-based capture of NEAT1-associated DNA. ( G ) Consensus motif in NEAT1-associated DNA sites (314 of top 500 peaks ranked by peak P -value). ( H ) TDF analysis predicting the triplex-forming potential of NEAT1 on ASO-captured DNA regions. Significant TFRs along NEAT1 are shown in orange, the number of target sites (DBS) for each TFR in purple. For TFR- and ASO-based capture assays nucleic acids isolated from HeLa S3 chromatin were used.

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Sequencing, Incubation, Labeling, One-tailed Test, MANN-WHITNEY, Allele-specific Oligonucleotide, Isolation

    Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Validation of triplex-forming RNA and DNAs. ( A ) TDF analysis predicting the potential of top 1000 enriched TriplexRNA (DNA-IP) regions (ranked by peak P -value) to bind to active promoters defined by ChromHMM. Number of TFRs in RNA (per kilobase of RNA, left) and the number of putative DBSs at promoters (per kilobase of RNA, right) are shown. Boxplot borders are defined by the 1st and 3rd quantiles of the distributions, the middle line corresponds to the median value. The top whisker denotes the maximum value within the third quartile plus 1.5 times the interquartile range (bottom whisker is defined analogously). Dark gray dots represent outliers with values higher or lower than whiskers. Further box plots are based on the same definitions. ( B ) Motif analysis of triplexes formed between TriplexRNA (DNA-IP) and active promoters. The diagram depicts the fraction of antiparallel and parallel triplexes with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( C ) TDF analysis comparing the triplex-forming potential of top 2000 TriplexDNA-seq regions with top 1000 TriplexRNA (DNA-IP) (ranked by peak P -value). The number of putative DBSs (per kilobase of RNA) is shown. ( D ) Motif analysis of predicted triplexes formed between TriplexRNAs (DNA-IP) and TriplexDNA. The diagram depicts the fraction of antiparallel and parallel triplexes, with the respective motif and nucleotide composition of TFRs in TriplexRNA. ( E ) Box plot classifying triplex interactions between TriplexRNAs (DNA-IP) and TriplexDNA-seq regions as cis ( > 10 kb in the same chromosome) and trans (at different chromosomes) interactions, excluding underrepresented local interactions (within 10 kb distance). ( F ) EMSAs using 10 or 100 pmol of synthetic TriplexRNAs and 0.25 pmol of double–stranded 32 P-labeled oligonucleotides comprising target regions from TriplexDNA ( Supplementary Table S2 ). Reactions marked with an asterisk (*) were treated with 0.5 U RNase H. As a control (C), RNA without a putative TFR was used. Potential Hoogsteen base pairing between motifs and respective TFR sequences are shown; mismatches are marked (*). TriplexRNA-seq and TriplexDNA-seq data are from HeLa S3 cells. Adjusted P -values

    Article Snippet: Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (NEB).

    Techniques: Whisker Assay, Labeling