Nebnext dna library prep master mix set

Manufactured by Illumina

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The NEBNext DNA Library Prep Master Mix Set is a collection of reagents designed for the preparation of DNA libraries for next-generation sequencing. It provides the necessary components for the enzymatic steps involved in the library preparation process, including DNA fragmentation, end repair, dA-tailing, and adapter ligation.

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Lab products found in correlation

Hiseq 2000, by Illumina (4 mentions) Hiseq 2500, by Illumina (3 mentions) Nebnext rna first strand synthesis module, by New England Biolabs (2 mentions) Mnase, by Takara Bio (2 mentions) Sureselect human all exon v5, by Agilent Technologies (2 mentions) Nebnext high fidelity pcr master mix, by New England Biolabs (2 mentions) E series, by Covaris (2 mentions) Herculase 2 fusion dna polymerase, by Agilent Technologies (2 mentions) Nextseq 500, by Illumina (2 mentions) Miseq system, by Illumina (2 mentions) Ovation rna seq system v2, by Tecan (2 mentions) Kapa library quantification kit, by Roche (2 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Hiseq 2500 platform, by Illumina (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Novaseq 6000, by Illumina (1 mentions) Kapa biosystems library quantification kit, by Roche (1 mentions) Bcl2fastq conversion software v1, by Illumina (1 mentions) Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)

Close spelling variants of this product

NEBNext DNA Library Prep Master Mix (5 citations) NEBNext DNA Library Prep Master Mix Set for Illumina kit (4 citations)

27 protocols using nebnext dna library prep master mix set

Sequencing of Bacterial DNA and RNA

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Before the DNA and RNA extraction, cells were starved for 2 days and ampicillin was added to reduce contamination by E. coli and other bacteria. Cells were harvested by centrifugation at 300 × g for 5 min. DNA was extracted using phenol chloroform extraction followed by ethanol precipitation. RNA extraction was performed with the RNeasy Plus minikit (Qiagen, Germany) following the manufacturer’s instructions.
The DNA library was constructed with NEBNext DNA library prep master mix set for Illumina (New England BioLabs [NEB], USA) following the manufacturer’s instructions. High-throughput sequencing was performed on an Illumina Hiseq 2500 platform. The paired-end sequencing produced 20G of clean data after quality control and removing adapters (read length: 150 bp).
The RNA libraries were generated using NEBNext Ultra RNA library prep kit for Illumina (NEB, USA) following the manufacturer’s instructions. Four RNA-seq replicates were sequenced. High-throughput sequencing was performed on Illumina Hiseq 2500 platform and produced 10G of clean data for each RNA library after quality control and removing adapters (read length: 150 bp).

Zheng W., Wang C., Lynch M, & Gao S. (2021). The Compact Macronuclear Genome of the Ciliate Halteria grandinella: A Transcriptome-Like Genome with 23,000 Nanochromosomes. mBio, 12(1), e01964-20.

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Paired-end Sequencing of Circular DNA

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We used 1ug amplified Circular DNA for paired-end (PE) library construction using the NEBNext DNA Library Prep Master Mix set for Illumina (New England Biolabs). PE high-throughput sequencing (150 cycles) was performed according to the manufacturer’s protocol (Illumina), and 150bp (PE150) on both ends of the library was sequenced by Illumina Novaseq 6000(Genedenovo Bio, Guangzhou, China).

Yang H., He J., Huang S., Yang H., Yi Q., Tao Y., Chen M., Zhang X, & Qi H. (2021). Identification and Characterization of Extrachromosomal Circular DNA in Human Placentas With Fetal Growth Restriction. Frontiers in Immunology, 12, 780779.

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Directional RNA Sequencing Library Prep

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For long RNA library preparation a Ribo-zero Magnetic Gold rRNA removal kit (Epicenter, IIlumina Inc.) was used to remove ribosomal RNA from the total RNA. First strand synthesis was performed using NEBNext RNA first strand synthesis module (New England BioLabs Inc., Ipswich, MA, USA). Immediately thereafter, directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this, cDNAs were used for standard library preparation protocol using NEBNext DNA Library Prep Master Mix Set for Illumina. End-repair was performed followed by polyA addition and custom adapter ligation. After ligation, material was individually barcoded with unique in-house genomics service lab primers. Library quality and concentration were assessed by a Qubit 2.0 Fluorometer and DNA 1000 chips on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-end sequencing was performed on all samples (100 bp paired-end directional reads). Raw reads were de-multiplexed using a bcl2fastq conversion software v1.8.3 (Illumina, Inc.) with default settings.

Jeppesen D.K., Fenix A.M., Franklin J.L., Higginbotham J.N., Zhang Q., Zimmerman L.J., Liebler D.C., Ping J., Liu Q., Evans R., Fissell W.H., Patton J.G., Rome L.H., Burnette D.T, & Coffey R.J. (2019). Reassessment of Exosome Composition. Cell, 177(2), 428-445.e18.

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ChIRP-seq Analysis of Mouse ES Cells

Cited 2 times

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The ChIRP-seq assay was performed as described previously^{50 (link)}. Mouse ES cells were cultured as above and treated with Dimethyl sulfoxide (DMSO) or 500nM JQ1^{48 (link)} (Gift from J. Bradner) for 1 hour at 37°C. Isolated RNA was used in qRT-PCR analysis (Stratagene) to quantify enrichment of 7SK and depletion of other cellular RNAs. Isolated DNA was used for qPCR analysis or to make deep sequencing libraries with the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Library DNA sequenced from a single end for 75 cycles on an Illumina HiSeq 2500 (Summary of all sequencing experiments are listed in Supplementary Table 5).
Sequencing reads were first trimmed of adaptors (FASTX Toolkit) and mapped using Bowtie to a custom bowtie index containing repetitive RNA elements (rRNA, snRNAs, and y-RNAs⁵¹). Subsequent reads were then mapped to mm9. Mapped reads were separately shifted towards the 3′ end using MACS and normalized to a total of 10 million reads. Even and Odd replicates were merged as described previously^{50 (link)} by taking the lower of the two read density values at each nucleotide across the entire genome. The full pipeline is available at https://github.com/bdo311/chirpseq-analysis.

Flynn R.A., Do B.T., Rubin A.J., Calo E., Lee B., Kuchelmeister H., Rale M., Chu C., Kool E.T., Wysocka J., Khavari P.A, & Chang H.Y. (2016). 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology, 23(3), 231-238.

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N. crassa Nucleosome Sequencing

Cited 1 time

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N. crassa cells were grown and digested with micrococcal nuclease as previously described (McKnight et al., 2021 (link)) with the following modifications. MNase (Takara) concentration was optimized for each strain to yield ~80%–90% mononucleosomes (20 units for N3752, N3753, N7966, N8018, N7990, N7992, N7988, and N7989; 40 units for N6877; 60 units for N4718; and 80 units for N4730, N6170, N6171, N6876, N8016, and N8017). All digestions were for 10 min at 37°C, RNase (40 µg) treatment was for 1.5 h at 42°C, and proteinase K (200 µg) treatment was for 1 hr at 65°C. About 10 µg of gel-purified mononucleosome DNA was prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Sequencing was performed by the University of Oregon Genomics and Cell Characterization Core Facility.

Wiles E.T., Mumford C.C., McNaught K.J., Tanizawa H, & Selker E.U. (2022). The ACF chromatin-remodeling complex is essential for Polycomb repression. eLife, 11, e77595.

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Exome Capture of Tumor and Blood Samples

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DNA from both tumor and peripheral blood samples were used in the exome capture protocol as previously described.^{34 (link)} Briefly, 200 ng DNA was fragmented on a Covaris E-Series using the settings suggested in the Sureselect^XT Target Enrichment System for Illumina Paired-End Sequencing Library protocol (version 1.5). DNA was end-repaired, A-tailed and adaptors ligated using the NEBNext DNA library prep master mix set for Illumina (New England Biolabs, Hitchin, UK). Adaptor-ligated DNA was amplified using the NEBNext High-fidelity PCR master mix (New England Biolabs) using 7 PCR cycles and 750 ng DNA hybridized against RNA baits overnight (SureSelect Human All Exon V5, Agilent Technologies). The RNA baits were designed against the human exome with the addition of custom baits that tiled the IGH, IGK, IGL and MYC loci in order to detect the major translocations in myeloma. After hybridisation the captured DNA was indexed and amplified using Herculase II fusion DNA polymerase (Agilent Technologies) for 8 PCR cycles. Four exome samples were pooled and run on one lane of a HiSeq 2000 (Illumina, Hinxton, UK) using 76-bp paired end reads.

Walker B.A., Wardell C.P., Murison A., Boyle E.M., Begum D.B., Dahir N.M., Proszek P.Z., Melchor L., Pawlyn C., Kaiser M.F., Johnson D.C., Qiang Y.W., Jones J.R., Cairns D.A., Gregory W.M., Owen R.G., Cook G., Drayson M.T., Jackson G.H., Davies F.E, & Morgan G.J. (2015). APOBEC family mutational signatures are associated with poor prognosis translocations in multiple myeloma. Nature communications, 6, 6997.

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Chromatin Immunoprecipitation of Anp32e in HeLa Cells

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HeLa cells stably expressing Flag-Anp32e were used for ChIP-Seq experiments; the cells were crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature, and the reaction was quenched by adding glycine at a final concentration of 0.15 M. The cells were washed with PBS and then resuspended in 20 mM Tris, pH 8.0, 300 mM NaCl and 0.1% SDS and sonicated to 200-to 500-bp chromatin fragments using a Bioruptor system (Diagenode). ChIP experiments were performed as previously reported^{41 (link)}. Sequencing libraries were generated according to Illumina's recommendations using the NEBNext DNA Library Prep Master Mix Set for Illumina. The libraries were single-end sequenced using HiSeq 2000.
Additional methods for crystal structure determination, analytical ultracentrifugation, recombinant protein IP, and bioinformatics analysis for Chip-Seq are given as the Supplementary information, Data S1.

Mao Z., Pan L., Wang W., Sun J., Shan S., Dong Q., Liang X., Dai L., Ding X., Chen S., Zhang Z., Zhu B, & Zhou Z. (2014). Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z. Cell Research, 24(4), 389-399.

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Amplification and Sequencing of eccDNA

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eccDNA was amplified using the REPLI-g Mini Kit (Qiagen, 150023), utilizing Multiple Displacement Amplification with random primers. EccDNA (≥ 20 ng) in 5 μL Buffer EB was denatured by incubation for 3 min with 5 μL Buffer D1, Denaturation was stopped by addition of 10 μL Buffer N1, after which 29 μL Repli-g Mini reaction buffer and 1 μL Repli-g Mini DNA polymerase were added and the reaction mixture incubated at 30°C for 16 hrs. After inactivating the polymerase at 65°C for 3 min, the buffer was exchanged using a Microcon YM-100 (Millipore, 42413) or DNA Fast Flow (Millipore, MRCF0R100) columns. 5 μg amplified DNA was used for paired-end library construction using the NEBNext DNA Library prep Master Mix set for Illumina (New England Biolabs, E6040S). Paired-end high-throughput sequencing (50 cycles) was performed according to the manufacturer’s protocol (Illumina) either on the Illumina Genome Analyzer IIX at the University of Virginia DNA Sciences Core (Charlottesville, VA, USA), the HiSeq2000 at BGI (Hong Kong), or the HiSeq2500 at HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA).

Kumar P., Dillon L.W., Shibata Y., Jazaeri A., Jones D.R, & Dutta A. (2017). Normal and Cancerous Tissues Release extrachromosomal circular DNA (eccDNA) into the Circulation. Molecular cancer research : MCR, 15(9), 1197-1205.

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Laser Capture Microdissection for Single-Cell RNA-Seq

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Tissues for LCM were assessed for their structure and integrity by Hematoxylin and Eosin (H&E) staining. Total RNA of whole tissue section was purified using mirVana miRNA Isolation Kit (Invitrogen). RNA integrity was assessed using the Bioanalyzer 2000 (Agilent). LCM staining protocol : cryosection were fixed in cold acetone and briefly air dried, immunofluorescence staining was performed on a HistoGene cold bloc (ThermoFisher) using modified immunofluorescence staining protocol with higher concentration of conjugated antibodies, all reagents included SUPERase inhibitor (ThermoFisher). After staining, samples were dehydrated in 75%, 95% and 100% Ethanol successively, the incubated 5 min in Xylene and air dried. Laser capture microdissection was performed on an Arcturus XT LCM with CapSure Macro LCM caps (ThermoFisher). After harvest, RNA was isolated with PicoPure RNA isolation kit following manufacturer protocol. SMART-Seq V1/V4 Ultra Low Input RNA Kit was used for cDNA synthesis and amplification. cDNAs were processed to the library preparation with NEBNext DNA Library Prep Master Mix Set for Illumina and 75bp single reads were used on Illumina NextSeq 500 or Novaseq sequencer (Illumina).

Martinek J., Lin J., Kim K.I., Wang V.G., Wu T.C., Chiorazzi M., Boruchov H., Gulati A., Seeniraj S., Sun L., Marches F., Robson P., Rongvaux A., Flavell R.A., George J., Chuang J.H., Banchereau J, & Palucka K. (2022). Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function. Cell Reports Medicine, 3(5), 100621.

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Illumina Sequencing Library Preparation

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Libraries were prepared using NEBNext DNA library prep master mix set for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. In brief, 1 μg of genomic DNA per sample was sheared to generate DNA fragments of 250–300 bp using the Covaris S2 AFA system (Covaris Inc. Woburn, MA, USA) and ligated to Illumina specific adaptors for multiplexing. Pre-capture amplified samples were pooled—10 samples for MiSeq and up to 23 samples for HiSeq 1500 sequencing—and hybridized to the customized in-solution capture library for 72 hours, subsequently eluted and post-capture amplified by ligation mediated (LM-) PCR. This amplified enriched DNA was used as input for direct cluster generation and sequencing on an Illumina MiSeq system (2 × 150 bp PE reads) or for cluster generation on a cBot system and sequencing in rapid mode (23 samples per lane, altogether up to 46 samples) on an Illumina HiSeq 1500 instrument (2 × 150 bp PE reads) (Illumina, San Diego, CA, USA). A more detailed protocol is available in online S1 Materials And Methods.

Eisenberger T., Decker C., Hiersche M., Hamann R.C., Decker E., Neuber S., Frank V., Bolz H.J., Fehrenbach H., Pape L., Toenshoff B., Mache C., Latta K, & Bergmann C. (2015). An Efficient and Comprehensive Strategy for Genetic Diagnostics of Polycystic Kidney Disease. PLoS ONE, 10(2), e0116680.

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