Nebnext ultra 2 directional kit

Manufactured by New England Biolabs

Sourced in United States

The NEBNext Ultra II Directional kit is a reagent system designed for the preparation of directional RNA-seq libraries from total RNA. The kit includes reagents and protocols for RNA fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, adapter ligation, and amplification.

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Lab products found in correlation

Nebnext poly a mrna magnetic isolation module, by New England Biolabs (3 mentions) Nextseq 500, by Illumina (2 mentions) Monarch total rna miniprep kit, by New England Biolabs (1 mentions) Rrna depletion kit, by New England Biolabs (1 mentions) Bioanalyzer small rna chip, by Agilent Technologies (1 mentions) Multiplex oligos, by New England Biolabs (1 mentions) Next rrna depletion kit, by New England Biolabs (1 mentions) Nextseq 550, by Illumina (1 mentions) Doxycycline, by Merck Group (1 mentions) Rna br assay kit, by Thermo Fisher Scientific (1 mentions) Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Streptavidin beads, by Thermo Fisher Scientific (1 mentions) Real time analysis software rta, by Illumina (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Hiseq 4000, by Agilent Technologies (1 mentions)

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10 protocols using nebnext ultra 2 directional kit

Transcriptomic Analysis of Granulosa-like Cells

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Total RNA was extracted from sorted FOXL2+ granulosa-like cells using the Arcturus PicoPure kit (Thermo Fisher), or from COV434 cells and hiPSCs using the Monarch Total RNA Miniprep kit (NEB). For experiments involving TF overexpression, TF expression plasmids were integrated into hiPSCs as described above (50 fmol/200,000 cells). After selection with puromycin, TF expression was induced using doxycycline (1000 ng/ml).
Two biological replicates were collected for each sample (iPSC, hiPSC+ individual TFs, sorted FOXL2+, no-TF differentiation, KGN, COV434). Libraries were prepared using the NEBNext Ultra II Directional kit following the manufacturer’s protocol, and sequenced on an Illumina NextSeq 500 (2 × 75 bp paired-end reads). The TPM data shown in Figure 3 were generated using kallisto (Bray et al., 2016 (link)) to pseudoalign reads to the reference human transcriptome (Ensembl GRCh38 v96). Differential expression analysis was performed using DESeq2. PantherDB (Mi et al., 2021 (link)) was used to calculate gene ontology enrichment for significantly upregulated (log₂fc >3, p_adj < 0.05) and downregulated (log₂fc <−3, p_adj < 0.05) genes for each sample relative to hiPSCs.

Pierson Smela M.D., Kramme C.C., Fortuna P.R., Adams J.L., Su R., Dong E., Kobayashi M., Brixi G., Kavirayuni V.S., Tysinger E., Kohman R.E., Shioda T., Chatterjee P, & Church G.M. (2023). Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression. eLife, 12, e83291.

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RNA Isolation and Sequencing Protocol

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RNA was isolated with TRIzol as described and DNase treated. When total RNA was sequenced, 1 microgram RNA was first depleted for ribosomal RNA using the rRNA Depletion Kit (NEB E6310). Total RNA libraries were then prepared using the NEBNext Ultra II Directional Kit (NEB E7760). Libraries were quantified with the KAPA Illumina Quantification Kit (KAPA KK4824).

Gutbrod M.J., Roche B., Steinberg J.I., Lakhani A.A., Chang K., Schorn A.J, & Martienssen R.A. (2022). Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4. Nature Communications, 13, 1001.

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mRNA-seq Library Preparation Protocol

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NEBNext Ultra II Directional kit (NEB, Cat. # E776) with the NEBNext poly(A) mRNA Magnetic isolation module (NEB, Cat. # E7490) was used to generate mRNA-seq libraries. Briefly, 400 ng of high-quality total RNA was used to purify poly(A) mRNA, fragmented, reverse-transcribed with random primers, adapter ligated, and amplified according to manufacturer recommendations. The final libraries were validated on the bioanalyzer, pooled, and sequenced on the NextSeq 500 using paired end 40 bp chemistry.

Dagar S., Sharma M., Tsaprailis G., Tapia C.S., Crynen G., Joshi P.S., Shahani N, & Subramaniam S. (2024). Ribosome Profiling and Mass Spectrometry Reveal Widespread Mitochondrial Translation Defects in a Striatal Cell Model of Huntington Disease. Molecular & Cellular Proteomics : MCP, 23(4), 100746.

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Fission Yeast RNA-seq and Differential Expression

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Libraries were constructed from immunoprecipitated RNA samples by the RNomics Platform at the Université de Sherbrooke in Sherbrooke, Quebec. RNA quality was assessed with a Bioanalyzer small RNA chip (Agilent, 5067-1548). Libraries were constructed with the NEBNext Ultra II Directional Kit (NEB, E7760S) and amplified with ten PCR cycles. cDNA libraries were sequenced on an Illumina NextSeq 500 with two runs per sample, each for 50-bp single-end reads. Following fastp processing (Version 0.20.1), reads from the first sequencing run were aligned to the fission yeast genome (ASM294v2) with Bowtie 2^{60 (link),61} and counted with featurecounts^{62 (link)} using the EF2 build of the ENSEMBL fission yeast genome. Differential expression analysis was performed using edgeR, with reads filtered to include transcripts with at least 1 count per million (CPM) in each sample, and libraries were normalized by Trimmed Mean of M-values (TMM)^{63 ,64 (link)}.

Porat J., El Baidouri M., Grigull J., Deragon J.M, & Bayfield M.A. (2022). The methyl phosphate capping enzyme Bmc1/Bin3 is a stable component of the fission yeast telomerase holoenzyme. Nature Communications, 13, 1277.

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RNA Purification and Sequencing Preparation

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The NEB Next rRNA depletion kit (E6310) was used for RNA purification, as several samples had RNA integrity numbers <8 (but >7). Most samples had RNA integrity numbers >8. After rRNA depletion, the NEBNext UltraII Directional Kit (E7765) and NEB Multiplex Oligos (Kits-E7335,E7710) were used to generate and barcode sample libraries. Library concentrations were quantified by quantitative polymerase chain reaction using KAPA Quant assays, and values were used to generate an equimolar pool to ensure equal distribution of reads across all samples.

Tipton A.E., Del Angel Y.C., Hixson K., Carlsen J., Strode D., Busquet N., Mesches M.H., Gonzalez M.I., Napoli E., Russek S.J, & Brooks-Kayal A.R. (2023). Selective neuronal knockout of STAT3 function inhibits epilepsy progression, improves cognition, and restores dysregulated gene networks in a temporal lobe epilepsy model. Annals of neurology, 94(1), 106-122.

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Directional RNA-seq Library Preparation

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Sequencing libraries of three biological replicates were prepared using NEBNext Ultra II Directional Kit (New England Biolabs, Ipswich, MA, USA) In brief, 200 to 300 ng total RNA was used as input in the polyA enrichment module protocol. Enriched samples were fragmented and transcribed into cDNA. Following universal adapter ligation, samples were barcoded using dual indexing primers. Samples were sequenced to 30 to 50 million single-end 75 bp reads on the Illumina Nextseq 550 sequencer (Illumina).

Kwik M., Hainzl S., Oppelt J., Tichy B., Koller U., Bernardinelli E., Steiner M., Zara G., Nofziger C., Weis S., Paulmichl M., Dossena S., Patsch W, & Soyal S.M. (2021). Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases. International Journal of Molecular Sciences, 22(7), 3296.

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Transcriptomic analysis of striatal cells

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Total RNA extracted from the cultured striatal cells as noted under Ribosome profiling were used for mRNA-seq library preparation. NEBNext Ultra II Directional kit (NEB, Cat. no. E776) with the NEBNext poly(A) mRNA magnetic isolation module (NEB, Cat. no. E7490) was used to generate mRNA-Seq libraries. Briefly, 400 ng of high-quality total RNA was used to purify poly(A) mRNA, fragmented, reverse-transcribed with random primers, adapter-ligated, and amplified according to the manufacturer’s recommendations. The final libraries were validated on the bioanalyzer, pooled, and sequenced on the NextSeq 500 using paired-end 40 bp chemistry.

Eshraghi M., Karunadharma P.P., Blin J., Shahani N., Ricci E.P., Michel A., Urban N.T., Galli N., Sharma M., Ramírez-Jarquín U.N., Florescu K., Hernandez J, & Subramaniam S. (2021). Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease. Nature Communications, 12, 1461.

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Transcriptome Analysis of Psoriatic Keratinocytes

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HaCaT_sh and HaCaT_ctrl cells were initially seeded in complete medium and 4 h later stimulated with doxycycline (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 24 h. Subsequently, the medium was replaced with fresh medium containing both doxycycline and pro-inflammatory psoriatic cytokines (IFNγ, TNFα, IL17) at concentrations of 20 ng/mL, 25 ng/mL, and 100 ng/mL, respectively. After 72 h of stimulation, the cells were lysed, and RNA was isolated directly from the plates using the RNEasy mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration of RNA was quantified using a Qubit fluorimeter with the RNA BR Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). For transcriptome sequencing, libraries were prepared using the NEBNext^® Ultra™ II Directional kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. The libraries were then mixed in equimolar amounts, and their quality was assessed using a BioAnalyzer 2100 microfluidic analyzer (Agilent Santa Clara, CA, USA) with the Agilent DNA High Sensitivity Kit (Agilent Santa Clara, CA, USA).

Zolotarenko A, & Bruskin S. (2024). IQGAP3 Is an Important Mediator of Skin Inflammatory Diseases. International Journal of Molecular Sciences, 25(8), 4545.

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NEBNext Ultra II Directional mRNA-seq

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NEBNext Ultra II Directional kit (NEB, Cat. # E776) with the NEBNext poly(A) mRNA Magnetic isolation module (NEB, Cat. # E7490) was used generate mRNA-seq libraries.
Briefly, 400ng of high-quality total RNA was used to purify poly(A) mRNA, fragmented, reverse-transcribed with random primers, adapter ligated, and amplified according to manufacturer recommendations. The final libraries were validated on the bioanalyzer, pooled, and sequenced on the NextSeq 500 using paired-end 40bp chemistry.

Subramaniam S., & Shahani N. (2021). Ribosome Profiling Reveals a Dichotomy Between Ribosome Occupancy of Nuclear-Encoded and Mitochondrial-Encoded OXPHOS mRNA Transcripts in a Striatal Cell Model of Huntington Disease.

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Exome-enriched RNA sequencing protocol

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Briefly, stranded RNAseq libraries were generated as per the manufacturer's recommendations, but without the transcriptome enrichment step (pre-capture libraries).
Transcriptome enrichment was achieved by the hybridization of the pre-capture library to the exome panels tested. Since the probe baits were biotinylated, hybridized libraries were captured using streptavidin beads (ThermoFisher, Waltham, MA) and PCR amplified-on-beads to generate a post-capture library. All post-capture libraries were subjected to quality control on an Agilent Bioanalyzer and normalized to 2nM. The postcapture libraries obtained from each capture platform were pooled, and each pool sequenced on one lane of a paired-end read flow cell for 2x100 cycles on a HiSeq4000 to obtain ~40M reads per sample. The primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). CASAVA 1.8.2 software was then used to demultiplex samples and generate raw reads and respective quality scores (Supplementary Tables 1 and2). Libraries were made as described above using the NEBNext Ultra II Directional kit (PN-E7760, New England Biolabs, Ipswich, MA). 5µg of pooled indexed libraries (500ng each)

Shohdy K.S., Bareja R., Sigouros M., Wilkes D., Dorsaint P., Manohar J., Bockelman D., Xiang J., Kim R., Mosquera J.M., Elemento O., Sboner A., Alonso A., & Faltas B.M. (2021). Functional Comparison of Different Exome Capture-based Methods for Transcriptomic Profiling of Formalin-Fixed Paraffin-Embedded Tumor Samples.

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