Ultra 2 directional rna library prep kit

Manufactured by Illumina

The Ultra II Directional RNA Library Prep Kit is a laboratory equipment product designed for preparing directional RNA libraries for next-generation sequencing. It provides a streamlined workflow for converting RNA samples into sequencing-ready libraries, preserving the original orientation of the RNA transcripts.

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Lab products found in correlation

Magnetic mrna isolation kit, by New England Biolabs (1 mentions) Novaseq, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Agilent small rna kit, by Agilent Technologies (1 mentions) Novaseq 6000, by Illumina (1 mentions) Nextseq 500 system, by Illumina (1 mentions) Nextseq 500 550 high output v2.5 kit, by Illumina (1 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions) Isogen, by Nippon Gene (1 mentions) Nextseq 500, by Illumina (1 mentions) Ampure bead, by Beckman Coulter (1 mentions) Novaseq 6000 sequencer, by Illumina (1 mentions) Ds 11 spectrophotometer, by DeNovix (1 mentions) Lineage cell depletion kit, by Miltenyi Biotec (1 mentions) Facs aria 2, by Miltenyi Biotec (1 mentions) Hiseq next generation sequencing instrument, by Illumina (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

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6 protocols using ultra 2 directional rna library prep kit

Illumina RNA-Seq Library Preparation

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RNA purity/quality (RIN ≥ 7) was assessed with the 2100 Agilent Bioanalyzer by using the Agilent small RNA kit. RNAseq involved using the Integragen platform. Libraries were prepared with the Ultra II Directional RNA Library Prep Kit for Illumina protocol according to the supplier recommendations. Briefly, the key stages of this protocol are successive for the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1 µg total RNA (with the Magnetic mRNA Isolation Kit from NEB), fragmentation by using divalent cations under elevated temperature to obtain approximately 300 bp pieces, double-strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing was then carried out on Paired End 100b reads of Illumina NovaSeq. Image analysis and base calling involved using Illumina Real Time Analysis v3.4.4 with default parameters.

Tchicaya-Bouanga J., Hung Y.J., Schwartz J.M., Yoon D.J., Chotard E., Marty C., Odri G.A., de Pinieux G., Cohen-Solal M, & Modrowski D. (2022). A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases. Cell Death & Disease, 13(9), 819.

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RNA-seq Library Construction and Analysis

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Library construction for RNA sequencing was conducted with the NEB Ultra II Directional RNA Library Prep kit (E7760), and sequencing was performed on an Illumina NovaSeq 6000 (50 bp paired-end) by the University of Calgary’s Centre for Health Genomics and Informatics facility. All samples passed quality checks with fastqc and were then aligned to the mouse genome (GRCm38/mm10) using STAR [18 (link)]. Count tables were assembled with htseq-count using the reverse strand and intersection non-empty mode, along with all other default settings [19 (link)]. The visualization of sample variance and differential expression analysis was completed on R console (version 3.4.3 and 4.1.0) (https://www.R-project.org/) and using the SeqMonk software (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) (accessed between 1 July 2019 and 31 July 2022). Prior to collapsing lanes, the intra-lane variability was assessed with centred-log ratio transformed scaled biplots. No lane replicates contained sufficiently high variance to be located in different PCA quadrants, and all lanes were then aggregated. Gene ontology analyses were performed with enrichR (https://maayanlab.cloud/Enrichr/), Panther (http://www.pantherdb.org/) and Metascape (https://metascape.org/) tools (accessed between 1 July 2019 and 30 June 2022).

Ballasy N.N., Bering E.A., Kokorudz C., Radford B.N., Zhao X., Dean W, & Hemberger M. (2022). Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells. Cells, 11(16), 2466.

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RNA-seq Analysis of LSK and CD8+ T-cells

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RNA was extracted from LSK and CD8⁺ T‐cells. Total RNA was extracted using ISOGEN (Nippon Gene). RNA sequencing was performed by the Liaison Laboratory Research Promotion Center (LILA), Kumamoto University. Library cDNAs were prepared using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and an Ultra II Directional RNA Library Prep Kit (Illumina) and sequenced using an Illumina NextSeq 500 system (Illumina) with a Nextseq 500/550 High Output v2.5 Kit (Illumina) to obtain single‐end 75‐nt reads. The resulting reads were aligned to the mouse genome UCSC mm10 using STAR ver.2.6.0a software after trimming to remove the adapter sequence and low‐quality ends using Trim Galore! V0.5.0 (cutadapt v1.16). Gene expression levels were measured as counts, and transcripts per million were determined using RSEM v1.3.1. A GTF file was derived from UCSC mm10. Gene Ontology analysis was performed using Gene Set Enrichment Analysis (GSEA) in the clusterProfiler package ver.4.1.0. GSEA software was downloaded from the websites of the Broad Institute (http://software.broadinstitute.org/gsea/downloads.jsp).

Kawano S., Araki K., Bai J., Furukawa I., Tateishi K., Yoshinobu K., Usuki S., Nimmo R.A., Kaname T., Yoshihara M., Takahashi S., Sashida G, & Araki M. (2023). A gain‐of‐function mutation in microRNA 142 is sufficient to cause the development of T‐cell leukemia in mice. Cancer Science, 114(7), 2821-2834.

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CUT&RUN for Histone Modifications and Chromatin Remodelers

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CUT&RUN was performed exactly as described in^{62 (link),63} using EZH2(Cell signaling #5246S), H3K27me3 (Active motif #61017) and ATRX antibodies. DNA was end-repaired using End-It Repair Kit, tailed with an A using Klenow exo minus, and ligated to adapters (NEB #E7600S) with T4 DNA ligase. Fragments >150 bp were size-selected with AMpure beads (Beckman Coulter) and subjected to ligation-mediated PCR amplification with barcoded adapters (NEB #E7600S) for Illumina sequencing using Q5 DNA polymerase. All enzymes except Q5 (NEB) were from Enzymatics (a Qiagen company). PolyA RNA (NEB # E7490S) was isolated from two biological replicates and libraries prepared using the Ultra II directional RNA library prep kit for illumina (NEB #E7760S). Sequencing was performed on a NextSeq 500 (Illumina).

Ren W., Medeiros N., Warneford-Thomson R., Wulfridge P., Yan Q., Bian J., Sidoli S., Garcia B.A., Skordalakes E., Joyce E., Bonasio R, & Sarma K. (2020). Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function. Nature Communications, 11, 2219.

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RNA Sequencing of Muscle Samples

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RNA was extracted from muscle samples using the TRIzol protocol^{112 (link)}. The integrity of the RNA was verified using a standard quality metric denominated RNA integrity number (RIN) value using the Agilent 4200 TapeStation system and the concentration was measured using the DeNovix DS-11 spectrophotometer. Five hundred nanograms of RNA were used to prepare the RNA sequencing libraries using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced using the Illumina NovaSeq 6000 sequencer. Reads were demultiplexed using bcl2fastq v. 2.20.0.

Walitt B., Singh K., LaMunion S.R., Hallett M., Jacobson S., Chen K., Enose-Akahata Y., Apps R., Barb J.J., Bedard P., Brychta R.J., Buckley A.W., Burbelo P.D., Calco B., Cathay B., Chen L., Chigurupati S., Chen J., Cheung F., Chin L.M., Coleman B.W., Courville A.B., Deming M.S., Drinkard B., Feng L.R., Ferrucci L., Gabel S.A., Gavin A., Goldstein D.S., Hassanzadeh S., Horan S.C., Horovitz S.G., Johnson K.R., Govan A.J., Knutson K.M., Kreskow J.D., Levin M., Lyons J.J., Madian N., Malik N., Mammen A.L., McCulloch J.A., McGurrin P.M., Milner J.D., Moaddel R., Mueller G.A., Mukherjee A., Muñoz-Braceras S., Norato G., Pak K., Pinal-Fernandez I., Popa T., Reoma L.B., Sack M.N., Safavi F., Saligan L.N., Sellers B.A., Sinclair S., Smith B., Snow J., Solin S., Stussman B.J., Trinchieri G., Turner S.A., Vetter C.S., Vial F., Vizioli C., Williams A., Yang S.B, & Nath A. (2024). Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome. Nature Communications, 15, 907.

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RNA-Seq Analysis of U2af1-Deleted Bone Marrow

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LSK cells from the BM of control and U2af1-deleted BMT mice, and lineage-negative (Lin^-) cells from the BM of control and primary U2af1-deleted mice were first enriched using Lineage cell depletion kit from Miltenyi and then sorted using a FACS Aria II at 7 days after pI-pC induction. Total RNA was extracted from BM LSK and Lin^- cells using RNeasy Mini kit (Qiagen). RNA sequencing was performed using Ultra II Directional RNA Library prep kit for Illumina (from NEB) and Hiseq next-generation sequencing instrument (Illumina). Gene set enrichment analysis was performed as described [19 ]. To identify splicing events, rMATS [20 ] was used. RT-PCR followed by gel electrophoresis was used to identify splicing events. Image J software was used to quantify the band intensity. Sequences of the primers are available in the supplemental Table 3. RNA-seq data from U2af1 cKO mice will be deposited to publicly available database (GEO). RNA-seq data for MP (Lin^-c-kit⁺) cells from U2af1 S34F knock-in mice: GSE112174.

Dutta A., Yang Y., Le B.T., Zhang Y., Abdel-Wahab O., Zang C, & Mohi G. (2021). U2af1 is required for survival and function of hematopoietic stem/progenitor cells. Leukemia, 35(8), 2382-2398.

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