nebnext ultra end repair da tailing module  (New England Biolabs)


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  • 99
    Name:
    NEBNext Ultra End Repair dA Tailing Module
    Description:
    NEBNext Ultra End Repair dA Tailing Module 96 rxns
    Catalog Number:
    E7442L
    Price:
    731
    Size:
    96 rxns
    Category:
    DNA Template Preparation for PCR
    Score:
    85
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    Structured Review

    New England Biolabs nebnext ultra end repair da tailing module
    NEBNext Ultra End Repair dA Tailing Module
    NEBNext Ultra End Repair dA Tailing Module 96 rxns
    https://www.bioz.com/result/nebnext ultra end repair da tailing module/product/New England Biolabs
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    nebnext ultra end repair da tailing module - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres"

    Article Title: Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres

    Journal: Genetics

    doi: 10.1534/genetics.115.177360

    Genes and repetitive DNA in centromeres. (A) Overlap between CENH3 enrichment and genetic elements. Within the centromere, we assigned each 20-kb locus into one of two categories, “low CENH3 loci” and “high CENH3 loci,” based on whether it exhibited less than or greater than the median CENH3 enrichment. Results are shown for three methods of measuring CENH3 enrichment. The first two are relative to a total nucleosome control produced by MNase digestion of total chromatin and relative to a randomly sheared naked DNA control produced by NEBNext dsDNA Fragmentase ( Gent et al. 2014 ). In both cases, only uniquely mapping reads were considered. In the third method, no control was used, and enrichment was defined by raw read counts, including nonuniquely mapping reads. Errors bars are standard errors of the means for each set of loci. (B) Comparison of genetic elements in centromeric 20-kb loci and whole-genome 20-kb loci, but without regard to relative CENH3 enrichment within centromeres
    Figure Legend Snippet: Genes and repetitive DNA in centromeres. (A) Overlap between CENH3 enrichment and genetic elements. Within the centromere, we assigned each 20-kb locus into one of two categories, “low CENH3 loci” and “high CENH3 loci,” based on whether it exhibited less than or greater than the median CENH3 enrichment. Results are shown for three methods of measuring CENH3 enrichment. The first two are relative to a total nucleosome control produced by MNase digestion of total chromatin and relative to a randomly sheared naked DNA control produced by NEBNext dsDNA Fragmentase ( Gent et al. 2014 ). In both cases, only uniquely mapping reads were considered. In the third method, no control was used, and enrichment was defined by raw read counts, including nonuniquely mapping reads. Errors bars are standard errors of the means for each set of loci. (B) Comparison of genetic elements in centromeric 20-kb loci and whole-genome 20-kb loci, but without regard to relative CENH3 enrichment within centromeres

    Techniques Used: Produced

    Related Articles

    Amplification:

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
    Article Snippet: Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L). .. The reaction was incubated at 25 °C overnight in a thermocycler.

    Article Title: Structural and spatial chromatin features at developmental gene loci in human pluripotent stem cells
    Article Snippet: The 3C libraries were sonicated into 300–700-bp-long fragments in microtubes (Covaris 520045) with Covaris E210 under the following conditions: duty cycle 5%, intensity 3, cycles per burst 200, and time 150 s. End-repairing and A-tailing were performed by using NEBNext Ultra End Repair/dA-Tailing Module (NEB E7442S) according to the manufacturer’s instructions. .. The adaptor-ligated 3C libraries were purified with 1.8× AMPureXP beads.

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads. .. Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols.

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments. .. Illumina multiplexing adapters were ligated to DNA fragments using a NEBNext Ultra Ligation Module for DNA (NEB, E7445) and purified with 1.2× Agencourt AMPure XP beads (Beckman, A63881).

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. A 20:1 molar excess of Duplex Sequencing adapters was ligated to the sample DNA using the NEBNext Ultra Ligation Module (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. .. DNA size selection was performed using 2% Agarose Gel; then 200~500 bp DNA fragments were excised and purified using Qiagen Gel Extraction Kit (Qiagen, Germany).

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The cDNA was amplified by using KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit) following the ONT 1D Kit’s manual. .. End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step.

    Article Title: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
    Article Snippet: To achieve the 1 μg of DNA needed for the Oxford Nanopore library prep, the full-length cDNA product was split into five aliquots and amplified for 13 cycles with KAPA Hifi Readymix 2 × (KAPA) using the ISPCR or multiplex cellular index primers. .. The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix).

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    DNA Ligation:

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: The library preparation for Oxford Nanopore sequencing was performed using the manufacturer’s recommended protocol—1D Genomic DNA by ligation for the SQK-LSK108 kit (Oxford Nanopore Technologies, Oxford, UK). .. Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA).

    Synthesized:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs). .. The direct RNA sequencing approach Libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA001; Oxford Nanopore Technologies).

    Blocking Assay:

    Article Title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes
    Article Snippet: Paragraph title: Recovery of Integration Sites Using PCR and a Blocking Primer ... DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences).

    Real-time Polymerase Chain Reaction:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Incubation:

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
    Article Snippet: Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L). .. The ligation mixture (30 μL) contains end repaired/A-tailed cfDNA (normally ranging between 1 pg to a few ng), HA (at 50:1 ratio against cfDNA), 1X ligation buffer, and 1uL NEB Quick LigationTM Kit (NEB, M2200L).

    Article Title: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing
    Article Snippet: DNA was fragmented using NEBNext dsDNA Fragmentase reagents, supplemented with 15 mM MgCl2 and incubated at 37°C for 30 min. .. Purified fragments were repaired and deoxyribosyladenine (dA) tailed using a NEBNext Ultra end repair/dA-tailing module.

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: The condition of incubation is 3 h at 60°C for BstUI and at 37°C for AciI and Hinp1I. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: DNA repair was performed using NEBNext FFPE Repair Mix (M6630, NEB). .. End-prep was performed using a NEBNext End repair/dA-tailing Module (E7546, NEB) and end-prepped DNAs were purified using Agencourt AMPure XP beads (Beckman Coulter).

    Modification:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The cDNA library was prepared using the Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies) according to the modified 1D strand switching cDNA by ligation protocol. .. End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step.

    Flow Cytometry:

    Article Title: Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
    Article Snippet: The Oxford Nanopore MinION Genomic DNA Sequencing Kit (R9 flow cell chemistry) was used to prepare the DNA libraries according to the manufacturer’s instructions. .. Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.).

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: End-prep was performed using a NEBNext End repair/dA-tailing Module (E7546, NEB) and end-prepped DNAs were purified using Agencourt AMPure XP beads (Beckman Coulter). .. Adapter ligation and tether attachment were conducted using the NEBNext Blunt/TA Ligase Master Mix (M0367S, NEB) and Ligation Sequencing Kit SQK-LSK208 for 2D, SQK-LSK108 for 1D and SQK-LSK308 for 1D2 .

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs). .. The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer (through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific).

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA). .. Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA).

    Article Title: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
    Article Snippet: The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix). .. The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix).

    Gas Chromatography:

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Positive Control:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments. .. A custom Nimblegen capture system (Roche) was designed to capture the genomic regions from 250 bp upstream to 250 bp downstream of each of the 10,673 selected cancer risk-associated SNPs using the online NimbleDesign Software with the default settings ( http://sequencing.roche.com/products/software/nimbledesign-software.html ).

    Ligation:

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
    Article Snippet: The T oligo has an extra ‘T’ at the 3′ end but has no 5′ phosphate. .. Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L). .. The ligation mixture (30 μL) contains end repaired/A-tailed cfDNA (normally ranging between 1 pg to a few ng), HA (at 50:1 ratio against cfDNA), 1X ligation buffer, and 1uL NEB Quick LigationTM Kit (NEB, M2200L).

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. A 20:1 molar excess of Duplex Sequencing adapters was ligated to the sample DNA using the NEBNext Ultra Ligation Module (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: It was also tested for using BstUI alone as RE2 for comparison. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. The sample obtained was purified and size selected through an E-gel run (Invitrogen, EX 2% Agarose) into three pieces, which were separately purified, and each piece was eluted in 20 μl of elution buffer.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: Primer sequences were showed in . .. For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. .. DNA size selection was performed using 2% Agarose Gel; then 200~500 bp DNA fragments were excised and purified using Qiagen Gel Extraction Kit (Qiagen, Germany).

    Article Title: Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells
    Article Snippet: The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. .. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). .. The DNA libraries were amplified for 15 cycles and subjected to deep sequencing with an Illumina HiSeq 2000.

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes
    Article Snippet: Samples of genomic DNA were prepared for Illumina sequencing by random shearing using a Covaris M220 ultrasonicator to achieve an average size distribution of 800–900 bp. .. DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences). .. Ligated DNA was split into at least four replicates prior to ligation-mediated (LM) PCR amplification (PCR1, 25 total cycles).

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The cDNA was amplified by using KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit) following the ONT 1D Kit’s manual. .. End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step. .. The barcoded- sample was amplified by PCR using KAPA HiFi DNA Polymerase, as well as the C11 PCR barcode according to the 1D PCR barcoding protocol.

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    MicroChIP Assay:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.

    Hemagglutination Assay:

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
    Article Snippet: Paragraph title: Ligation of HA to DNA fragments ... Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L).

    Generated:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads. .. Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols.

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: End-prep was performed using a NEBNext End repair/dA-tailing Module (E7546, NEB) and end-prepped DNAs were purified using Agencourt AMPure XP beads (Beckman Coulter). .. Libraries were then purified using MyOne C1 beads (65001, Thermo Fisher Scientific) and sequenced for 48 hours by MinION Mk 1B with the SpotON Flow Cell (FLO-MIN106, R9.4 version for 2D; FLO-MIN107, R9.5 version for 1D and 1D2 , Oxford Nanopore Technologies).

    DNA Sequencing:

    Article Title: Closed Genome Sequence of Phytopathogen Biocontrol Agent Bacillus velezensis Strain AGVL-005, Isolated from Soybean
    Article Snippet: To gain insight into the use of Bacillus velezensis strain AGVL-005 (a Gram-positive and rod-shaped soil bacterium) for the biological control of phytopathogens, we sequenced the genome using the MinION Mk1B device (MIN-101B; Oxford Nanopore Technologies, UK). .. Briefly, ∼1 µg of unsheared genomic DNA was submitted to end repair and dA-tailing steps using the NEBNext Ultra end repair/dA-tailing module (New England BioLabs, USA) and then treated with the 1D Genomic DNA sequencing kit for the MinION device (catalog number SQK-LSK-108; Oxford Nanopore Technologies, UK). .. The resulting library was sequenced using a flow cell Spot-ON Mk1 (FLO-MIN 106 R9 version; Oxford Nanopore Technologies), with the R9 version library loading bead kit (EXP-LLB001; Oxford Nanopore Technologies).

    Article Title: Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
    Article Snippet: The Oxford Nanopore MinION Genomic DNA Sequencing Kit (R9 flow cell chemistry) was used to prepare the DNA libraries according to the manufacturer’s instructions. .. Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.).

    Sequencing:

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Paragraph title: Duplex Sequencing ... Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions.

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: It was also tested for using BstUI alone as RE2 for comparison. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. The sample obtained was purified and size selected through an E-gel run (Invitrogen, EX 2% Agarose) into three pieces, which were separately purified, and each piece was eluted in 20 μl of elution buffer.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. .. For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively.

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Article Title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes
    Article Snippet: Samples of genomic DNA were prepared for Illumina sequencing by random shearing using a Covaris M220 ultrasonicator to achieve an average size distribution of 800–900 bp. .. DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences).

    Article Title: Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
    Article Snippet: Paragraph title: MinION library preparation, sequencing and alignment ... Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.).

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: Paragraph title: Validation by physical long-read sequencing of MinION ... End-prep was performed using a NEBNext End repair/dA-tailing Module (E7546, NEB) and end-prepped DNAs were purified using Agencourt AMPure XP beads (Beckman Coulter).

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: Paragraph title: Oxford Nanopore MinION cDNA sequencing and barcoding ... End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step.

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: Paragraph title: Oxford Nanopore MinION sequencing ... End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA). .. The adapted library was purified using Agencourt AMPure XP (Beckman Coulter Inc.) and applied to a primed FLO-MIN106 R9.4 SpotON Flow Cell attached to MinION (Oxford Nanopore Technologies).

    Article Title: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
    Article Snippet: To achieve the 1 μg of DNA needed for the Oxford Nanopore library prep, the full-length cDNA product was split into five aliquots and amplified for 13 cycles with KAPA Hifi Readymix 2 × (KAPA) using the ISPCR or multiplex cellular index primers. .. The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix). .. Ligation reaction was performed using Blunt/TA ligase master mix (NEB).

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    Sonication:

    Article Title: Structural and spatial chromatin features at developmental gene loci in human pluripotent stem cells
    Article Snippet: For qPCR, 100 ng of template was used for each reaction, and the reaction steps were 1 cycle at 95 °C for 30 s and 45 cycles at 95 °C for 5 s, and 1 cycle at 60 °C for 30 s. Interaction frequencies were normalized to the DNA amount based on measurements using the GAPDH locus. .. The 3C libraries were sonicated into 300–700-bp-long fragments in microtubes (Covaris 520045) with Covaris E210 under the following conditions: duty cycle 5%, intensity 3, cycles per burst 200, and time 150 s. End-repairing and A-tailing were performed by using NEBNext Ultra End Repair/dA-Tailing Module (NEB E7442S) according to the manufacturer’s instructions. .. Ligation of the Splinkerette adaptor for sequencing by the next-generation sequencer (Top: 5′-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′, Bottom: 3′-phosphate-GATCGGAAGAGCTGTTTTTTTTTTCAAAAAAA-5′) to the A-tailed 3C libraries was performed with Ligation high Ver.2 (TOYOBO LGK-201) at 16 °C for 30 min and 10:1 adapter:library molar ratio.

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Seventy-five microliters of 1× Hank's buffered salt solution was added, and the lysate was sonicated in 1.5-mL TPX microtubes (Diagenode). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: Genomic DNA (1 μg) from each of the ten individuals of the Chinese Han population were pooled and sheared into ~ 500-bp fragments by sonication (Covaris S220). .. End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments.

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Duplex Sequencing was performed as previously described ( ; ) with several modifications. .. Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. A 20:1 molar excess of Duplex Sequencing adapters was ligated to the sample DNA using the NEBNext Ultra Ligation Module (New England Biolabs).

    Binding Assay:

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: It was also tested for using BstUI alone as RE2 for comparison. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. The sample obtained was purified and size selected through an E-gel run (Invitrogen, EX 2% Agarose) into three pieces, which were separately purified, and each piece was eluted in 20 μl of elution buffer.

    ChIP-sequencing:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads. .. Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: Paragraph title: 2.9. Library Preparation and ChIP-Seq ... For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively.

    Article Title: Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells
    Article Snippet: Paragraph title: ChIP-Seq ... After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB).

    DNA Extraction:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: Human saliva was collected using a saliva DNA Sample Collection Kit (ZEESAN, 401002) and genomic DNA was isolated using a genomic DNA extraction kit (ZEESAN, 602001). .. End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments.

    RNA Sequencing Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Paragraph title: RNA-seq analysis ... Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs). .. Samples were loaded on R9.4 SpotON Flow Cells, and base calling was performed using Albacore v1.2.6.

    Magnetic Beads:

    Article Title: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing
    Article Snippet: The fragmentation reaction was stopped by adding 5 μl of 0.5 M EDTA (Ambion, Foster City, CA), and fragments were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA). .. Purified fragments were repaired and deoxyribosyladenine (dA) tailed using a NEBNext Ultra end repair/dA-tailing module.

    Article Title: Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells
    Article Snippet: The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. .. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB).

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step. .. The PCR product was end-repaired, then it was followed by adapter ligation using the sequencing adapters supplied in the kit and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Isolation:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: Human saliva was collected using a saliva DNA Sample Collection Kit (ZEESAN, 401002) and genomic DNA was isolated using a genomic DNA extraction kit (ZEESAN, 602001). .. End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments.

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. A 20:1 molar excess of Duplex Sequencing adapters was ligated to the sample DNA using the NEBNext Ultra Ligation Module (New England Biolabs).

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Size-exclusion Chromatography:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Chromatin was sheared using a Bioruptor (Diagenode) with five active cycles (30 sec on, 30 sec off). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.

    Purification:

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
    Article Snippet: Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L). .. The reaction was incubated at 25 °C overnight in a thermocycler.

    Article Title: Structural and spatial chromatin features at developmental gene loci in human pluripotent stem cells
    Article Snippet: The 3C libraries were sonicated into 300–700-bp-long fragments in microtubes (Covaris 520045) with Covaris E210 under the following conditions: duty cycle 5%, intensity 3, cycles per burst 200, and time 150 s. End-repairing and A-tailing were performed by using NEBNext Ultra End Repair/dA-Tailing Module (NEB E7442S) according to the manufacturer’s instructions. .. Ligation of the Splinkerette adaptor for sequencing by the next-generation sequencer (Top: 5′-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′, Bottom: 3′-phosphate-GATCGGAAGAGCTGTTTTTTTTTTCAAAAAAA-5′) to the A-tailed 3C libraries was performed with Ligation high Ver.2 (TOYOBO LGK-201) at 16 °C for 30 min and 10:1 adapter:library molar ratio.

    Article Title: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing
    Article Snippet: The fragmentation reaction was stopped by adding 5 μl of 0.5 M EDTA (Ambion, Foster City, CA), and fragments were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA). .. Purified fragments were repaired and deoxyribosyladenine (dA) tailed using a NEBNext Ultra end repair/dA-tailing module. .. NEBNext adaptor for Illumina was ligated to the dA-tailed fragments using the NEBNext Ultra ligation module, and the adaptor was linearized by uracil-specific excision reagent enzyme in accordance with the manufacturer's instructions.

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads. .. Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols.

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: DNA fragments between 450 and 500 bp were size-selected on a 1.2% high-resolution agarose gel and recovered by TIANgel midi purification kit (TIANGEN, DP209). .. End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments.

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: It was also tested for using BstUI alone as RE2 for comparison. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. The sample obtained was purified and size selected through an E-gel run (Invitrogen, EX 2% Agarose) into three pieces, which were separately purified, and each piece was eluted in 20 μl of elution buffer.

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Article Title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes
    Article Snippet: DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences). .. DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences).

    Article Title: Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
    Article Snippet: The Oxford Nanopore MinION Genomic DNA Sequencing Kit (R9 flow cell chemistry) was used to prepare the DNA libraries according to the manufacturer’s instructions. .. Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.). .. The DNA library entailing adaptor ligation and purification of double-stranded DNA with hairpin adaptor was prepared using the Nanopore Sequencing Kit SQK-NSK007 (R9 version).

    Article Title: Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines
    Article Snippet: DNA repair was performed using NEBNext FFPE Repair Mix (M6630, NEB). .. End-prep was performed using a NEBNext End repair/dA-tailing Module (E7546, NEB) and end-prepped DNAs were purified using Agencourt AMPure XP beads (Beckman Coulter). .. Adapter ligation and tether attachment were conducted using the NEBNext Blunt/TA Ligase Master Mix (M0367S, NEB) and Ligation Sequencing Kit SQK-LSK208 for 2D, SQK-LSK108 for 1D and SQK-LSK308 for 1D2 .

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step. .. The PCR product was end-repaired, then it was followed by adapter ligation using the sequencing adapters supplied in the kit and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA). .. Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA).

    Polymerase Chain Reaction:

    Article Title: Structural and spatial chromatin features at developmental gene loci in human pluripotent stem cells
    Article Snippet: The 3C libraries were sonicated into 300–700-bp-long fragments in microtubes (Covaris 520045) with Covaris E210 under the following conditions: duty cycle 5%, intensity 3, cycles per burst 200, and time 150 s. End-repairing and A-tailing were performed by using NEBNext Ultra End Repair/dA-Tailing Module (NEB E7442S) according to the manufacturer’s instructions. .. The adaptor-ligated 3C libraries were purified with 1.8× AMPureXP beads.

    Article Title: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing
    Article Snippet: Fragment libraries were prepared from approximately 50 ng of PCR products using NEBNext DNA library prep reagents for Illumina (New England BioLabs, Ipswich, MA) per the manufacturer's instruction. .. Purified fragments were repaired and deoxyribosyladenine (dA) tailed using a NEBNext Ultra end repair/dA-tailing module.

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments. .. Illumina multiplexing adapters were ligated to DNA fragments using a NEBNext Ultra Ligation Module for DNA (NEB, E7445) and purified with 1.2× Agencourt AMPure XP beads (Beckman, A63881).

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. A 20:1 molar excess of Duplex Sequencing adapters was ligated to the sample DNA using the NEBNext Ultra Ligation Module (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. .. For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively.

    Article Title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes
    Article Snippet: Paragraph title: Recovery of Integration Sites Using PCR and a Blocking Primer ... DNA fragment ends were repaired (5′ phosphorylated and dA tailed) prior to TA ligation with custom linkers using NEBNext Ultra End Repair/dA-Tailing and NEBNext Ultra Ligation Modules, respectively (see for linker design and for sequences).

    Article Title: Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
    Article Snippet: The Oxford Nanopore MinION Genomic DNA Sequencing Kit (R9 flow cell chemistry) was used to prepare the DNA libraries according to the manufacturer’s instructions. .. Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.). .. The DNA library entailing adaptor ligation and purification of double-stranded DNA with hairpin adaptor was prepared using the Nanopore Sequencing Kit SQK-NSK007 (R9 version).

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The cDNA was amplified by using KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit) following the ONT 1D Kit’s manual. .. End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step. .. The barcoded- sample was amplified by PCR using KAPA HiFi DNA Polymerase, as well as the C11 PCR barcode according to the 1D PCR barcoding protocol.

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: PCR was carried out using Kapa HiFi DNA polymerase (Kapa Biosystems) and the primers supplied in the kit. .. End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    Immunoprecipitation:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: Immunoprecipitation and washes were done as described in the True MicroChIP protocol, with elution using SPRI beads (1:1 ratio; Fisher Scientific, 09-981-123). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads. .. Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols.

    Article Title: Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity
    Article Snippet: Primer sequences were showed in . .. For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. .. DNA size selection was performed using 2% Agarose Gel; then 200~500 bp DNA fragments were excised and purified using Qiagen Gel Extraction Kit (Qiagen, Germany).

    Article Title: Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells
    Article Snippet: The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. .. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB).

    Construct:

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    cDNA Library Assay:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The cDNA library was prepared using the Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies) according to the modified 1D strand switching cDNA by ligation protocol. .. End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step.

    Article Title: Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus
    Article Snippet: The ‘strand switching cDNA by ligation ’ approach The cDNA library was prepared using the Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies) following the 1D strand switching cDNA by ligation protocol. .. End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Mouse Assay:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Chromatin Immunoprecipitation:

    Article Title: Dynamic changes in histone modifications precede de novo DNA methylation in oocytes
    Article Snippet: For immunoprecipitation, the following antibodies and amounts of antibody were used for both 25,000- and 5000-cell ChIP: 0.25 µg of H3K4me3 (Diagenode, pAb-003-050), 0.25 µg of H3K4me2 (Abcam, ab32356), 5 µL of H3K36me3 (donated by Robert Klose), and 0.25 µg of H3 (Abcam, ab1791). .. ChIP-seq libraries were generated from immunoprecipitated DNA using the NEBNext Ultra end repair/dA-Tailing module (E7742) followed by SPRI purification (1:1 ratio) and 15–20 cycles of amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.

    Article Title: Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells
    Article Snippet: The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. .. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). .. The DNA libraries were amplified for 15 cycles and subjected to deep sequencing with an Illumina HiSeq 2000.

    Plasmid Preparation:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments. .. Illumina multiplexing adapters were ligated to DNA fragments using a NEBNext Ultra Ligation Module for DNA (NEB, E7445) and purified with 1.2× Agencourt AMPure XP beads (Beckman, A63881).

    Software:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments. .. Adapter-ligated DNA fragments were amplified by PCR with amplification primers containing both illumina adapter sequences and homology arms with the vector (forward primer, GTAATAATTCTAGAGTCGGGGCGGGcatgAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; reverse primer, TATCATGTCTGCTCGAAGCGGCAtaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) using NEBNext® High-Fidelity 2× PCR Master Mix (NEB, M0541L) and purified with 1.2× Agencourt AMPure XP beads.

    Article Title: Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress
    Article Snippet: Briefly, 500 ng of total DNA was sonicated in 60 μl of nuclease-free ddH2 O and subjected to end-repair and 3′-dA-tailing using the NEBNext Ultra End-Repair/dA-Tailing Module (New England Biolabs) according to the vendor's instructions. .. The captured DNA samples were then sequenced on an Illumina HiSeq2500 using 101 bp paired-end sequencing.

    Article Title: Closed Genome Sequence of Phytopathogen Biocontrol Agent Bacillus velezensis Strain AGVL-005, Isolated from Soybean
    Article Snippet: Briefly, ∼1 µg of unsheared genomic DNA was submitted to end repair and dA-tailing steps using the NEBNext Ultra end repair/dA-tailing module (New England BioLabs, USA) and then treated with the 1D Genomic DNA sequencing kit for the MinION device (catalog number SQK-LSK-108; Oxford Nanopore Technologies, UK). .. Briefly, ∼1 µg of unsheared genomic DNA was submitted to end repair and dA-tailing steps using the NEBNext Ultra end repair/dA-tailing module (New England BioLabs, USA) and then treated with the 1D Genomic DNA sequencing kit for the MinION device (catalog number SQK-LSK-108; Oxford Nanopore Technologies, UK).

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA). .. The adapted library was purified using Agencourt AMPure XP (Beckman Coulter Inc.) and applied to a primed FLO-MIN106 R9.4 SpotON Flow Cell attached to MinION (Oxford Nanopore Technologies).

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification.

    Multiplex Assay:

    Article Title: Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing
    Article Snippet: Purified fragments were repaired and deoxyribosyladenine (dA) tailed using a NEBNext Ultra end repair/dA-tailing module. .. NEBNext adaptor for Illumina was ligated to the dA-tailed fragments using the NEBNext Ultra ligation module, and the adaptor was linearized by uracil-specific excision reagent enzyme in accordance with the manufacturer's instructions.

    Article Title: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
    Article Snippet: To achieve the 1 μg of DNA needed for the Oxford Nanopore library prep, the full-length cDNA product was split into five aliquots and amplified for 13 cycles with KAPA Hifi Readymix 2 × (KAPA) using the ISPCR or multiplex cellular index primers. .. The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix). .. Ligation reaction was performed using Blunt/TA ligase master mix (NEB).

    Selection:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: In brief, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). .. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module.

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification. .. The library quality was assessed with a Qubit 2.0 fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.

    Agarose Gel Electrophoresis:

    Article Title: Systematic identification of regulatory variants associated with cancer risk
    Article Snippet: DNA fragments between 450 and 500 bp were size-selected on a 1.2% high-resolution agarose gel and recovered by TIANgel midi purification kit (TIANGEN, DP209). .. End-repair and dA-tailing were performed with a NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442) with all recovered DNA fragments.

    Next-Generation Sequencing:

    Article Title: Essential role of the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells
    Article Snippet: Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. .. For strand-specific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-N-glycosylase (New England Biolabs) as described followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems).

    Concentration Assay:

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min. .. One-fourth of each piece of eluted DNA was applied to library PCR reaction (6–8 cycles) in 50 μl of reaction volume using Phusion high fidelity PCR master mix with GC buffer.

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: End repair was carried out on the samples using NEBNext End repair / dA-tailing Module (New England Biolabs), which was followed by “barcoding”: the C11 barcode (ONT PCR Barcoding Kit 96; EXP-PBC096) was ligated to the sample according to the 1D PCR barcoding (96) genomic DNA (SQK-LSK108) protocol, Barcode Adapter ligation step. .. The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter).

    Fractionation:

    Article Title: Dynamics of Heterotrophic Bacterial Assemblages within Synechococcus Cultures
    Article Snippet: DNA extractions from Synechococcus culture samples, without size fractionation, were used for amplification of the bacterial hypervariable V4 region of 16S rRNA gene using the primers 520F (5′-AYTGGGYDTAAAGNG-3′) and 802R (5′-TACNVGGGTATCTAATCC-3′) ( ). .. Sequencing libraries were constructed using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, USA), according to the manufacturer's recommendations in terms of NEBNext end prep, adaptor ligation, size selection, or cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA, and cleanup of PCR amplification.

    Nanopore Sequencing:

    Article Title: Utilization of Hybrid Assembly Approach to Determine the Genome of an Opportunistic Pathogenic Fungus, Candida albicans TIMM 1768
    Article Snippet: Paragraph title: Library Preparation and Oxford Nanopore Sequencing ... Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA).

    Article Title: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
    Article Snippet: Paragraph title: Nanopore sequencing ... The single cDNA or multiplex product was further end-repaired and dA-tailed using NEBNext Ultra End Repair/dA tailing mix (NEB), and adaptor ligated using the sequencing adaptors provided by ONT (HP Adaptor/Adaptor Mix).

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  • 99
    New England Biolabs nebnext end repair module
    Genes and repetitive DNA in centromeres. (A) Overlap between CENH3 enrichment and genetic elements. Within the centromere, we assigned each 20-kb locus into one of two categories, “low CENH3 loci” and “high CENH3 loci,” based on whether it exhibited less than or greater than the median CENH3 enrichment. Results are shown for three methods of measuring CENH3 enrichment. The first two are relative to a total nucleosome control produced by MNase digestion of total chromatin and relative to a randomly sheared naked DNA control produced by <t>NEBNext</t> dsDNA Fragmentase ( Gent et al. 2014 ). In both cases, only uniquely mapping reads were considered. In the third method, no control was used, and enrichment was defined by raw read counts, including nonuniquely mapping reads. Errors bars are standard errors of the means for each set of loci. (B) Comparison of genetic elements in centromeric 20-kb loci and whole-genome 20-kb loci, but without regard to relative CENH3 enrichment within centromeres
    Nebnext End Repair Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs 3 end
    LITE-Seq analysis. a Germline tissue used in this study. Whole gonads were isolated from adult hermaphrodite (XX) and male (XO) worms. Female germline samples were generated by manual removal of the spermatheca. Developmentally staged samples were obtained by microdissection of the distal mitotic proliferation zone, the meiotic pachytene region, and cellularized oocytes. b LITE-Seq protocol. Extracts of total RNA spiked with ERCC RNA standards were reverse transcribed using an anchored hairpin polydT primer. cDNAs were PCR amplified using a biotinylated half-hairpin primer, fragmented, and subjected to <t>3′-end</t> capture with streptavidin beads. Samples were barcoded, pooled, and subjected to paired-end sequencing using the standard Illumina Read 1 primer and a custom Read 2 primer that initiates sequencing immediately upstream of the polyA tail. c Mapping and quantification of transcript 3′ ends. Paired-end reads were mapped to the C. elegans reference genome (WS250) and assigned to genes with overlapping exons or to the closest gene within 1500 nt upstream. Representative 3′ CPA sites (arrowheads) and isoform abundance were determined by clustering 3′ end reads within ±12 nucleotides of the position with the most supported reads
    3 End, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ultra rna library prep kit
    sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from <t>RNA-seq</t> show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from <t>ESP</t> and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.
    Ultra Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Genes and repetitive DNA in centromeres. (A) Overlap between CENH3 enrichment and genetic elements. Within the centromere, we assigned each 20-kb locus into one of two categories, “low CENH3 loci” and “high CENH3 loci,” based on whether it exhibited less than or greater than the median CENH3 enrichment. Results are shown for three methods of measuring CENH3 enrichment. The first two are relative to a total nucleosome control produced by MNase digestion of total chromatin and relative to a randomly sheared naked DNA control produced by NEBNext dsDNA Fragmentase ( Gent et al. 2014 ). In both cases, only uniquely mapping reads were considered. In the third method, no control was used, and enrichment was defined by raw read counts, including nonuniquely mapping reads. Errors bars are standard errors of the means for each set of loci. (B) Comparison of genetic elements in centromeric 20-kb loci and whole-genome 20-kb loci, but without regard to relative CENH3 enrichment within centromeres

    Journal: Genetics

    Article Title: Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres

    doi: 10.1534/genetics.115.177360

    Figure Lengend Snippet: Genes and repetitive DNA in centromeres. (A) Overlap between CENH3 enrichment and genetic elements. Within the centromere, we assigned each 20-kb locus into one of two categories, “low CENH3 loci” and “high CENH3 loci,” based on whether it exhibited less than or greater than the median CENH3 enrichment. Results are shown for three methods of measuring CENH3 enrichment. The first two are relative to a total nucleosome control produced by MNase digestion of total chromatin and relative to a randomly sheared naked DNA control produced by NEBNext dsDNA Fragmentase ( Gent et al. 2014 ). In both cases, only uniquely mapping reads were considered. In the third method, no control was used, and enrichment was defined by raw read counts, including nonuniquely mapping reads. Errors bars are standard errors of the means for each set of loci. (B) Comparison of genetic elements in centromeric 20-kb loci and whole-genome 20-kb loci, but without regard to relative CENH3 enrichment within centromeres

    Article Snippet: We end-repaired and 5′-phosphorylated using the End Repair Module (New England Biolabs #E6050L), A-tailed with Klenow exo- (New England Biolabs #M0212S), and ligated to adapters (8.3 nM) with a Quick Ligase Kit (New England Biolabs #M2200S).

    Techniques: Produced

    LITE-Seq analysis. a Germline tissue used in this study. Whole gonads were isolated from adult hermaphrodite (XX) and male (XO) worms. Female germline samples were generated by manual removal of the spermatheca. Developmentally staged samples were obtained by microdissection of the distal mitotic proliferation zone, the meiotic pachytene region, and cellularized oocytes. b LITE-Seq protocol. Extracts of total RNA spiked with ERCC RNA standards were reverse transcribed using an anchored hairpin polydT primer. cDNAs were PCR amplified using a biotinylated half-hairpin primer, fragmented, and subjected to 3′-end capture with streptavidin beads. Samples were barcoded, pooled, and subjected to paired-end sequencing using the standard Illumina Read 1 primer and a custom Read 2 primer that initiates sequencing immediately upstream of the polyA tail. c Mapping and quantification of transcript 3′ ends. Paired-end reads were mapped to the C. elegans reference genome (WS250) and assigned to genes with overlapping exons or to the closest gene within 1500 nt upstream. Representative 3′ CPA sites (arrowheads) and isoform abundance were determined by clustering 3′ end reads within ±12 nucleotides of the position with the most supported reads

    Journal: Genome Biology

    Article Title: Developmental dynamics of gene expression and alternative polyadenylation in the Caenorhabditis elegans germline

    doi: 10.1186/s13059-017-1369-x

    Figure Lengend Snippet: LITE-Seq analysis. a Germline tissue used in this study. Whole gonads were isolated from adult hermaphrodite (XX) and male (XO) worms. Female germline samples were generated by manual removal of the spermatheca. Developmentally staged samples were obtained by microdissection of the distal mitotic proliferation zone, the meiotic pachytene region, and cellularized oocytes. b LITE-Seq protocol. Extracts of total RNA spiked with ERCC RNA standards were reverse transcribed using an anchored hairpin polydT primer. cDNAs were PCR amplified using a biotinylated half-hairpin primer, fragmented, and subjected to 3′-end capture with streptavidin beads. Samples were barcoded, pooled, and subjected to paired-end sequencing using the standard Illumina Read 1 primer and a custom Read 2 primer that initiates sequencing immediately upstream of the polyA tail. c Mapping and quantification of transcript 3′ ends. Paired-end reads were mapped to the C. elegans reference genome (WS250) and assigned to genes with overlapping exons or to the closest gene within 1500 nt upstream. Representative 3′ CPA sites (arrowheads) and isoform abundance were determined by clustering 3′ end reads within ±12 nucleotides of the position with the most supported reads

    Article Snippet: While still attached to beads, the captured cDNA was end-repaired and an adenosine was added to the 3′ end (NEBNext Ultra End Repair/dA-Tailing module).

    Techniques: Isolation, Generated, Laser Capture Microdissection, Polymerase Chain Reaction, Amplification, Sequencing

    sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.

    Journal: Nucleic Acids Research

    Article Title: The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978

    doi: 10.1093/nar/gky603

    Figure Lengend Snippet: sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.

    Article Snippet: Libraries of the ESP RNA samples were prepared using the NEBNext™ Ultra RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions.

    Techniques: Sequencing, RNA Sequencing Assay, Expressing, Northern Blot, Isolation, End-sequence Profiling, Software