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 (94 mentions) Trizol reagent, by Thermo Fisher Scientific (62 mentions) Hiseq 2500, by Illumina (51 mentions) Rna nano 6000 assay kit, by Agilent Technologies (41 mentions) Novaseq 6000, by Illumina (41 mentions) Rneasy mini kit, by Qiagen (38 mentions) Hiseq platform, by Illumina (36 mentions) Bioanalyzer 2100 system, by Agilent Technologies (33 mentions) Truseq pe cluster kit v3 cbot hs, by Illumina (32 mentions) Trizol, by Thermo Fisher Scientific (32 mentions) Novaseq platform, by Illumina (31 mentions) Agilent bioanalyzer 2100 system, by Agilent Technologies (30 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (29 mentions) Nanodrop, by Thermo Fisher Scientific (28 mentions) 2100 bioanalyzer, by Agilent Technologies (27 mentions) Nanophotometer spectrophotometer, by Implen (26 mentions) Hiseq 2500 platform, by Illumina (26 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (23 mentions) Hiseq 4000, by Illumina (23 mentions) Ampure xp system, by Beckman Coulter (20 mentions)

Close spelling variants of this product

NEBNext Ultra II Directional RNA Library Prep Kit (244 citations) NEBNext Ultra Directional RNA Library Prep Kit (217 citations) NEBNext Ultra II RNA Library Prep Kit (111 citations) NEBNext® Ultra™ RNA Library Prep Kit for Illumina® Kit (12 citations) NEBNext Ultra RNA Library Kit (9 citations) NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (9 citations) NEBNext Ultra small RNA Sample Library Prep Kit (8 citations) NEBNext RNA library prep kit (6 citations) NEBNext R UltraTM RNA Library Prep Kit for Illumina R (6 citations) NEBNext Ultra Library Prep Kit (5 citations)

606 protocols using nebnext ultra rna library prep kit

RNA-seq Library Preparation and Bioinformatic Analysis

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The library construction, sequencing and data analysis were all conducted by Novogene in Beijing. A total of 1.5 μg of RNA was used for library construction using the NEBNext^® Ultra TM RNA Library Prep Kit from Illumina^® (NEB, Ipswich, MA, USA). Sequencing libraries were generated using the NEBNext^® UltraTM RNA Library Prep Kit from Illumina^® (NEB, Ipswich, MA, USA), following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.
Clean data were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. The obtained clean data were spliced with Trinity (v2.6.6) software. Transcript functions were annotated based on the following databases: NR (NCBI non-redundant protein sequences); NT (NCBI non-redundant nucleotide sequences); Pfam (Protein family); KOG/COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database); GO (Gene Ontology).

Xiang L., Liu X., Shi Y., Li Y., Li W., Li F, & Chen K. (2021). Comparative Transcriptome Analysis Revealed Two Alternative Splicing bHLHs Account for Flower Color Alteration in Chrysanthemum. International Journal of Molecular Sciences, 22(23), 12769.

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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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RNA Extraction and RNA-Seq Library Preparation

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RNA was extracted from 100 mg leaf material using the RNeasy Plant Mini kit according to the manufacturer’s instructions (Qiagen, Germany). The quantity and quality of extracted RNA were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA-Seq libraries were prepared from 3 µg of each purified RNA with NEBNext UltraTM RNA Library Prep Kit of Illumina (NEB, USA) according to the manufacturer’s instructions. Briefly, the fragmentation of mRNA was enriched from total RNA by Oligo (dT) beads, and double-stranded cDNAs were synthesized. Libraries were purified using the AMPure XP system (Beckman Coulter, USA), and quantified using the Agilent 2100 Bioanalyzer. The pooled libraries were subjected to cluster generation and sequencing was carried out on the Illumina NovaSeq 6000 platform (250-bp, paired-end reads).

Li P., Xiao X., Wang J., Niu F., Huang J., Xie B., Ye L., Zhang C., Wang D., Wu Q., Zheng X., Gai Y., Li H, & Jiao C. (2023). Transcriptional insights of citrus defense response against Diaporthe citri. BMC Plant Biology, 23, 614.

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Integrated Genomic Analysis Pipeline

Cited 5 times

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DNA and RNA were extracted from tissue samples using AllPrep DNA/RNA Mini Kit(Qiagen). Exome libraries were constructed using the Agilent SureSelect Human All Exon V6. RNA sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit from Illumina® (NEB, USA) following the manufacturer’s recommendations. Both exome and RNA sequencing were conducted at Novogene Co., Ltd. with 150 bp paired-end reads.
After quality control which includes trimming adapters and removing low-quality reads, we mapped short reads to the hg19 reference genome using BWA MEM (v0.7.17)^{46 (link)}. Duplicated reads were subsequently marked using MarkDuplicates and base quality scores were recalibrated using GATK4 (v4.1.3.0)^{47 (link)}. Somatic variants were called using Mutect2^{48 (link)}. Variants were further filtered using GATK FilterMutectCalls with default parameters and were annotated with Oncotator (v1.9.9.0)^{49 (link)}. Somatic copy number variations as well as tumor purity were inferred using Sequenza (v3.0.0)^{50 (link)}. The genome instability index (GII) is defined as the proportion of the genome with abs(log2(CN/2)) > 0.2, where CN is the copy number of the focal genomic region^{51 (link)}.

Liu X., Zhang K., Kaya N.A., Jia Z., Wu D., Chen T., Liu Z., Zhu S., Hillmer A.M., Wuestefeld T., Liu J., Chan Y.S., Hu Z., Ma L., Jiang L, & Zhai W. (2024). Tumor phylogeography reveals block-shaped spatial heterogeneity and the mode of evolution in Hepatocellular Carcinoma. Nature Communications, 15, 3169.

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RNA-Seq Analysis of hiPSC-CMs in DMD

Cited 1 time

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RNA-sequencing was performed by GENEWIZ company using the Illumina HiSeq instrument. Total RNA from day 30 WT and DMD hiPSC-CMs were isolated using the TRIzol Reagent (Life Technologies). One microgram total RNA (RIN value above 7) was used for library construction using the NEBNext^® Ultra^TM RNA Library Prep Kit for Illumina^®.
Libraries were multiplexed and loaded onto Illumina HiSeq instrument (Illumina). Sequencing was carried out using a 2 × 150 bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. Differential expression analysis was carried out using the DESeq Bioconductor package (Anders and Huber, 2010 (link)). After adjusting with Benjamini and Hochberg’s approach for accounting the false discovery rate, P-value of p < 0.05 was deemed statistically significant and was used to identify differential expressed genes.

Li B., Xiong W., Liang W.M., Chiou J.S., Lin Y.J, & Chang A.C. (2021). Targeting of CAT and VCAM1 as Novel Therapeutic Targets for DMD Cardiomyopathy. Frontiers in Cell and Developmental Biology, 9, 659177.

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Transcriptomic Analysis of Rabbit IUA Treated with hUCB-MSCs

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The rabbit IUA samples with and without treated with hUCB-MSCs were used for transcriptome sequencing (3 vs 3). Firstly, total RNAs were extracted by Trizol method, RNA purity was detected by spectrophotometer, and RNA integrity was analyzed by agarose gel electrophoresis and Agilent 2100 BioAnalyzer. The Library was constructed using Illumina’s NEBNext® UltraTM RNA Library Prep Kit. Then, Illumina platform was used for library sequencing and 150 bp paired terminal reading was generated to obtain the sequence information of the fragment to be measured. After quality control and sequence alignment based on reference genome, DESeq2 software [24 (link)] was used to analyze the differentially expressed genes (DEGs) between the two groups. Finally, the DEGs were used for gene enrichment analysis based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).

Hua Q., Zhang Y., Li H., Li H., Jin R., Li L., Xiang Y., Tian M., Wang J., Sun L, & Wang Y. (2022). Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-κB signaling in rabbit intrauterine adhesions endometrium. Stem Cell Research & Therapy, 13, 301.

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Transcriptome Analysis of Primary Orbital Fibroblasts

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The total RNA of primary orbital fibroblasts was extracted using an RNA Purification Kit (Yishan Biotechnology, Shanghai, China). RNA extracts were analyzed using an RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA) in combination with the Bioanalyzer 2100 system. RNA sample preparations were carried out with a total amount of 1 μg RNA per sample. NEBNext^® UltraTM RNA Library Prep Kit (Illumina, CA, USA) was used for library preparation and index codes were added to each sample as attribute specifier sequence. After sample clustering, the library preparations were sequenced on an Illumina Novaseq platform. Quality control raw reads were processed using in-house Perl scripts. The reference genome index was built using Hisat2 (v2.0.5) and the paired-end reads were aligned against the reference genome using Hisat2 (v2.0.5) as well. The mapped reads of each sample were assembled by StringTie (v1.3.3b) (29 (link)). Differential expression testing was performed using the DESeq2 R package (1.20.0). Genes with a P-value < 0.05 as identified by DESeq2 were considered differentially expressed.

Wu P., Lin B., Huang S., Meng J., Zhang F., Zhou M., Hei X., Ke Y., Yang H, & Huang D. (2022). IL-11 Is Elevated and Drives the Profibrotic Phenotype Transition of Orbital Fibroblasts in Thyroid-Associated Ophthalmopathy. Frontiers in Endocrinology, 13, 846106.

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Transcriptome Analysis of COVID-19 Patients

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Total RNA was extracted from nucleated cells in whole blood. RNA purity was checked using NanoPhotometer spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s instructions. The library quality was determined on the Agilent Bioanalyzer 2100 system. Sequencing was performed on an Illumina Novaseq platform. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Differential expression analysis of two conditions (COVID-19 versus healthy) was performed using the DESeq2 R package (1.16.1).

Zhou Y., Liao X., Song X., He M., Xiao F., Jin X., Xie X., Zhang Z., Wang B., Zhou C., Kang Y, & Zhang W. (2021). Severe Adaptive Immune Suppression May Be Why Patients With Severe COVID-19 Cannot Be Discharged From the ICU Even After Negative Viral Tests. Frontiers in Immunology, 12, 755579.

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Transcriptome Analysis of Mammary Gland

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Mammary glands were minced and digested by collagenase and hyaluronidase to remove fat tissue, and red blood cells were removed by NH₄Cl solution. Total RNA was extracted from mammary epithelial cells which isolated from mouse mammary glands (four control and four cKO mice) at pregnancy day 14.5 and then sent to Novogene Co. Ltd. for library construction and sequencing. RNA quality was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, United States). Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina. The qualities of clean reads were assessed using FastQC (v0.11.5) and then the reads were mapped to mouse genome (GRCm38) using Hisat2 (v2.0.5). FPKM were calculated, and differential expression analysis was performed using DESeq2 (v1.16.1). Differentially expressed genes (p < 0.05) were chosen for further analysis. The RNA-sequencing data have been submitted to the GEO repository GSE164550.

Shao C., Lou P., Liu R., Bi X., Li G., Yang X., Sheng X., Xu J., Lv C, & Yu Z. (2021). Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland. Frontiers in Cell and Developmental Biology, 9, 691050.

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