Nebnext ultra rna library prep kit

Manufactured by Illumina

Sourced in United States, China, Germany, United Kingdom, Switzerland, Japan

The NEBNext Ultra RNA Library Prep Kit is a lab equipment product designed for the preparation of RNA libraries for next-generation sequencing. It provides a streamlined workflow for the conversion of RNA into a cDNA library suitable for sequencing on Illumina platforms.

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Lab products found in correlation

Agilent 2100 bioanalyzer, by Agilent Technologies (70 mentions) Trizol reagent, by Thermo Fisher Scientific (48 mentions) Hiseq 2500, by Illumina (46 mentions) Rneasy mini kit, by Qiagen (34 mentions) Novaseq 6000, by Illumina (33 mentions) Rna nano 6000 assay kit, by Agilent Technologies (26 mentions) Hiseq platform, by Illumina (26 mentions) Truseq pe cluster kit v3 cbot hs, by Illumina (24 mentions) Bioanalyzer 2100 system, by Agilent Technologies (24 mentions) Trizol, by Thermo Fisher Scientific (23 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (22 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (21 mentions) Hiseq 2500 platform, by Illumina (20 mentions) Novaseq platform, by Illumina (19 mentions) Hiseq 4000, by Illumina (19 mentions) 2100 bioanalyzer, by Agilent Technologies (18 mentions) Agilent bioanalyzer 2100 system, by Agilent Technologies (17 mentions) Nanophotometer spectrophotometer, by Implen (16 mentions) Nebnext poly a mrna magnetic isolation module, by Illumina (16 mentions) Hiseq 2000, by Illumina (14 mentions)

469 protocols using nebnext ultra rna library prep kit

mRNA Sequencing Library Preparation

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Messenger RNA (mRNA) was enriched with a NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490, Ipswich, MA). Using the enriched mRNA as template, cDNA libraries were constructed using the NEBNext Ultra RNA Library Prep Kit Illumina (NEB, E7530, Ipswich, MA) and NEBNext Multiplex Oligos for Illumina (Index Primer 1–12) (NEB, E7600, Ipswich, MA) following the manufacturer’s instructions. To verify the quality, DNA concentration and product size of the cDNA libraries, a Qubit 2.0 Fluorometer (Life Technologies, Q32866), Qubit dsDNA BR assay kit (Life Technologies, Q32850), High Sensitivity DNA Analysis Kit (Agilent, 5067–4626) and Bioanalyzer were used. cDNA libraries were sequenced on an Illumina MiSeq Platform employing a 150 base pair single-end NGS setting. Data from MiSeq sequencing runs were uploaded and stored in BaseSpace (https://basespace.illumina.com) for data analysis.

Ballesteros C., Tritten L., O’Neill M., Burkman E., Zaky W.I., Xia J., Moorhead A., Williams S.A, & Geary T.G. (2016). The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi. PLoS Neglected Tropical Diseases, 10(1), e0004311.

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Illumina-based mRNA Sequencing Protocol

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RNA concentration and purity were measured using the Qubit RNA BR Assay Kit (Life Technologies, Q10210, Burlington, ON) on an Agilent 2100 Bioanalyzer (Santa Clara, CA). mRNA was obtained by Poly (A) magnetic isolation (NEBNext Poly (A) mRNA Magnetic Isolation Module, NEB, Ipswich, MA). The enriched mRNA served as template for cDNA library preparation with the NEBNext^® Ultra RNA Library Prep Kit Illumina (NEB, E7530) and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (NEB, E7600) following the manufacturer's instructions. Assessment of quality, DNA concentration and product size of the cDNA was performed for each library using a Qubit^® 2.0 Fluorometer (Life Technologies, Q32866), Qubit^® dsDNA BR assay kit (Life Technologies, Q32850), High Sensitivity DNA Analysis Kit (Agilent, 5067-4626) and Agilent 2100 Bioanalyzer. cDNA libraries were sequenced on an Illumina MiSeq Platform employing a 150 base pair paired-end NGS setting.

O'Neill M., Ballesteros C., Tritten L., Burkman E., Zaky W.I., Xia J., Moorhead A., Williams S.A, & Geary T.G. (2016). Profiling the macrofilaricidal effects of flubendazole on adult female Brugia malayi using RNAseq. International Journal for Parasitology: Drugs and Drug Resistance, 6(3), 288-296.

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Transcriptomic Profiling of DLBCL

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Total RNA with sufficient quantity and quality was extracted using the RNeasy Mini Kit (Qiagen) from fresh-frozen tumor tissues of 162 patients among the 188 DLBCL. Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina. RNA sequencing was further conducted on an Illumina NovaSeq 6000 platform (Illumina). Detailed information on RNA sequencing and qPCR is provided in the online supplemental methods.

Zhang T., Liu H., Jiao L., Zhang Z., He J., Li L., Qiu L., Qian Z., Zhou S., Gong W., Meng B., Ren X., Zhang H, & Wang X. (2022). Genetic characteristics involving the PD-1/PD-L1/L2 and CD73/A2aR axes and the immunosuppressive microenvironment in DLBCL. Journal for Immunotherapy of Cancer, 10(4), e004114.

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RNA Extraction, mRNA Isolation, and Library Preparation

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Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. After quality control using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, United States), 1–2 µg of RNA was used to extract mRNA according to the NEB Next Poly(A) mRNA Magnetic Isolation Module. The RNA sequencing libraries were then constructed according to the manufacturer’s instructions for the Illumina NEBNext Ultra RNA Library Prep Kit (NEB). The generated libraries were pooled and sequenced on an Illumina NovaSeq™ 6000 following the vendor’s recommended protocol platforms with a 150-bp paired-end mode (sequenced by LC-Bio Technology Co., Ltd. Hangzhou, China).

Liu M., Zhao L., Wang Z., Su H., Wang T., Yang G., Chen L., Wu B., Zhao G., Guo J., Yang Z., Zhang J., Hao C., Ma T., Song Y., Bao S., Zuo Y., Li X, & Cao G. (2021). Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition. Frontiers in Cell and Developmental Biology, 9, 785055.

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Comprehensive Transcriptome Analysis of HepG2 Cells

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Total RNA was isolated from HepG2 using the Trizol Reagent (Ambion, USA) according to the manufacturer’s protocol, The quantity and integrity of RNA yield was assessed by using the K5500 (BeijingKaiao, China) and the Agilent 2200 TapeStation (Agilent Technologies, USA) separately. Briefly, the mRNA was enriched by oligodT conjugated NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, USA) according to instructions. And then fragmented to be approximately 200 bp. Subsequently, the RNA fragments were subjected to first strand and second strand cDNA synthesis following by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit (Thermo Fisher Scientific, USA). The libraries were sequenced using Illumina (Illumina, USA) with paired-end 150 bp at Ribobio Co. Ltd (Ribobio, China).

Liu X., Jin Y., Wan X., Liang X., Wang K., Liu J., Jiang J., Meng B., Han S., Zhou L., Cai S, & Zou F. (2022). SALIS transcriptionally represses IGFBP3/Caspase-7-mediated apoptosis by associating with STAT5A to promote hepatocellular carcinoma. Cell Death & Disease, 13(7), 642.

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Transcriptome Sequencing and Annotation

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Transcriptome sequencing was conducted by Novogene and sequencing libraries were generated using NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^®. Clustering of samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS. The library preparations were made on an Illumina Hiseq platform and paired-end reads were generated. The reads were de novo assembled and estimated using the Trinity pipeline. Gene functions were annotated based on data from the NCBI [34 (link)] Nr and Nt databases and from Pfam [35 (link)], the COG/KOG database [36 (link)], Swiss-Prot [37 (link)], the KEGG database [38 (link)], and the GO database [39 (link)].

Bu J., Zhang X., Li Q., Ma Y., Hu Z., Yang J., Liu X., Wang R., Jiao X., Chen T., Lai C., Cui G., Tang J., Kong Y., Yang L., Lin S., Chen Y., Guo J, & Huang L. (2022). Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids. Horticulture Research, 9, uhac152.

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Bovine Muscle Transcriptome Profiling

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The loin samples collected after slaughter were introduced in cryogenic tubes, frozen in liquid nitrogen and stored at −80°C until analysis. The RiboPure™ High‐Quality RNA Purification kit (Ambion) was used to extract total RNA, following the manufacturer's recommendations. NanoDrop equipment (NanoDrop Technologies) was used to quantify the RNA and Agilent 2100 Bioanalyzer device (Agilent Technologies) was used to measure RNA integrity (RNA integrity number). The values obtained for all the samples were higher than 8.
NEBNext^® Ultra™ RNA Library Prep Kit (Illumina) was used to build the paired end libraries for each sample. Novaseq 6000 sequence analyzer (Illumina Inc) to carry out multiplex sequencing of the libraries, with four samples per lane at Novogene (Novogene UK Company Limited), according to the manufacturer's instructions. Pair end reads of 150 bp were generated. The raw sequence data of the 12 animals has been deposited in the Gene Expression Omnibus database with the accession number: GSE178915.

Fernández‐Barroso M.Á., García‐Casco J.M., Núñez Y., Ramírez‐Hidalgo L., Matos G, & Muñoz M. (2022). Understanding the role of myoglobin content in Iberian pigs fattened in an extensive system through analysis of the transcriptome profile. Animal Genetics, 53(3), 352-367.

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RNA-seq analysis of skih-2 and nonu-1 in C. elegans

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25–50 day 1 adults were picked from a blank plate into S-basal solution and washed to remove E. coli. Animals were dissolved in trizol and total RNA extracted. Ribosomal RNA was depleted from 250 ng of total RNA using an NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and libraries were constructed using an NEBNext Ultra RNA Library Prep Kit for Illumina sequencing. Libraries were sequenced at the University of California, Santa Cruz using the Illumina NextSeq platform.
RNA-seq reads were trimmed with cutadapt and mapped using STAR (version 2.5.0a) to the C. elegans genome (WBCel235) with the unc-54 locus modified to match the unc-54(cc4092) allele. Reads that mapped within the annotated bounds of a protein coding gene were assigned to that gene. Multiply-mapping reads or reads that could not be unambiguously assigned to a gene (e.g., due to overlapping genes) were discarded. Read counts were median-normalized using DESeq (Anders and Huber, 2010 (link)).
For differential expression of endogenous mRNAs in skih-2 and nonu-1, genes with mRNAs that increased in biological duplicates (skih-2) or triplicates (nonu-1) were identified with DESeq. mRNA levels were deemed significantly different if they exhibited an adjusted p value < 0.05 (skih-2) or < 0.001 (nonu-1). Varying these cutoffs changed the number genes identified as skih-2 or nonu-1 targets, but did not alter our conclusions.

Glover M.L., Burroughs A.M., Monem P.C., Egelhofer T.A., Pule M.N., Aravind L, & Arribere J.A. (2020). NONU-1 Encodes a Conserved Endonuclease Required for mRNA Translation Surveillance. Cell reports, 30(13), 4321-4331.e4.

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Thoracic and Lumbar Disc RNA-seq

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Entire intervertebral discs from the thoracic and lumbar spine (T8-L5) of the P20 Col2Cre; Adgrg6^f/f and control mice were isolated in cold PBS, snap frozen and pulverized in liquid nitrogen. Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analyzed on Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). RNA fragments were reverse-transcribed to create the final cDNA libraries following the NEBNext Ultra RNA Library Prep Kit (Illumina, San Diego, USA), paired-end sequencing was performed. All raw reads are available as GSE128402 in the NCBI Gene Expression Omnibus.

Liu Z., Easson G.W., Zhao J., Makki N., Ahituv N., Hilton M.J., Tang S.Y, & Gray R.S. (2019). Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice. PLoS Genetics, 15(10), e1008096.

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Splenic CD4+ T cell activation and RNA-seq

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Splenic CD4⁺ T cells were activated with anti-CD3 antibody (5 μg/mL) and anti-CD28 antibody (2 μg/mL) in the presence or absence DMXAA (1 μg/mL) under neutral conditions for 2 days. Total RNA was extracted by TRIzol, chloroform, and isopropanol. RNA was quantified and qualified by NanoDrop (Thermo Fisher Scientific, Waltham, MA), agarose gel electrophoresis, and Agilent 2100 (Agilent, Santa Clara, CA). The library was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina at Novogene (Sacramento, CA). Qualified libraries were sequenced using a paired-end 150 run on an Illumina NovaSeq Platform (Illumina, San Diego, CA). Data were analyzed by Novogene and visualized by iDEP.⁴⁹ (link)

Yang W., Yu T., Zhou G., Yao S., Wakamiya M., Hu H., Paessler S., Sun J, & Cong Y. (2023). Intrinsic STING Switches off Pathogenetic Programs of Th1 Cells to Inhibit Colitis. Cellular and Molecular Gastroenterology and Hepatology, 15(5), 1161-1179.

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