Oligo dt 25 beads

Manufactured by New England Biolabs

Sourced in United States

Oligo(dT)25 beads are a type of magnetic bead-based product used for the isolation and purification of polyadenylated (poly(A)+) RNA from biological samples. The beads are coated with 25 thymidine (dT) nucleotides, which bind to the poly(A) tails of mRNA molecules, allowing them to be captured and separated from the rest of the RNA and cellular components.

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Lab products found in correlation

Hiseq 4000 platform, by Illumina (3 mentions) Murine rnase inhibitor, by New England Biolabs (2 mentions) Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions) Edta free protease inhibitor, by Roche (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Lambda exonuclease, by New England Biolabs (1 mentions) Multiplex oligos, by New England Biolabs (1 mentions) Dnase 1, by Promega (1 mentions) Nebnext ultra dna library prep kit, by Illumina (1 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Hiseq 2000 platform, by Illumina (1 mentions) Nextseq 500 system, by Illumina (1 mentions) Rotor gene 6000 pcr system, by Qiagen (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Dnase 1, by New England Biolabs (1 mentions) Smartscribe reverse transcriptase, by Takara Bio (1 mentions) Ampure beads, by Thermo Fisher Scientific (1 mentions) Phire 2, by Thermo Fisher Scientific (1 mentions) 2100 bioanalyzer, by Illumina (1 mentions) Hiseq 2500 platform, by Illumina (1 mentions)

Spelling variants of this product

Oligo-dT beads (18 citations) Oligo dT (10 citations) NEBNext Oligo(dT)25 beads (10 citations)

10 protocols using oligo dt 25 beads

Leishmania mRNA Interactome Capture

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For the mRNA interactome capture experiments, all conditions were equivalent for the Leishmania lifecycle forms PCF, META, AMA(MØ), and AMA(LD) between XL and nonXL samples except the irradiation. In vivo UV-crosslinking was performed using the LT40 “Minitron” system (UV03 Ltd.) (24 (link)). Cells at 5 × 10⁶ cells/ml confluence (∼0.6 OD) were irradiated for 120s (Fig. 2B; ∼1.6 mJ/cm²) for optimal in vivo RBP:RNA (mRNP) crosslinking with superior mRNA integrity and negligible heat stress compared with a standard Stratalinker which exposes the cells to 100× the heat (∼150 mJ/cm²) (24 (link)). Both AMA(MØ) and AMA (LD) were irradiated in host MØs, which were then homogenized to release parasites for gradient purification as above.
Triplicate samples of ∼5 × 10⁹ cells of each lifecycle stage (XL and nonXL) were resuspended in 15 ml Lysis buffer (25 (link)) (20 mm Tris-HCl, pH7.5, 500 mm LiCl, 0.5% LDS, 1 mm EDTA and 5 mm DTT with cOmplete EDTA-free Protease Inhibitors ^TM (Roche)) for 10 min at 0 °C, passed through a 25G needle until clear, centrifuged 10 min at 4000 × g and incubated with oligo(dT)₂₅ beads 30 min at 4 °C (New England Biolabs). Remaining steps for poly(A) RNA isolation were performed as described (18 (link)). mRNA-bound proteins were precipitated via TCA precipitation, and the resulting protein concentration measured using Micro BCA Protein assay kit (Thermo).

de Pablos L.M., Ferreira T.R., Dowle A.A., Forrester S., Parry E., Newling K, & Walrad P.B. (2019). The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite. Molecular & Cellular Proteomics : MCP, 18(7), 1271-1284.

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Puromycin-mRNA Ligation Optimization

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Purified mRNA was ligated to the puromycin linker, Nb_Disp_Puro using a DNA splint, Nb-GFP_Disp_Splint or HuSdL_Disp_Splint (Table II). For each 60 µL reaction, 5 µg mRNA was first annealed to 2.67 µM splint and 2.67 µM puromycin linker in the presence of 1× T4 ssRNA ligase buffer. Following annealing, 1 mM ATP, 1 µL murine RNase inhibitor (New England Biolabs, NEB), 3 µL T4 ssRNA Ligase were added and the reaction was incubated at room temperature overnight. Unligated linker and splint were cleaved using 5 µL lambda exonuclease (NEB) in a total volume of 240 µL, containing 24 µL of lambda exonuclease buffer, and subsequent incubation at 37°C for 45 mins. After the addition of 60 μL of 1 M Tris-HCl, pH 7.5, the enzyme was inactivated by incubation at 65°C for 5 mins and the reaction was placed on ice. The mRNA-puromycin linker ligation product was purified using 70 µL magnetic OligodT₂₅ beads (NEB), as per the manufacturer's instructions, and eluted twice in 30 uL (15 µL × 2) of 10 mM Tris-HCl, pH 7.5. The ligation efficiency of this method was typically between ~40–60%.

Doshi R., Chen B.R., Vibat C.R., Huang N., Lee C.W, & Chang G. (2014). In vitro nanobody discovery for integral membrane protein targets. Scientific Reports, 4, 6760.

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Transcriptomic Analysis of Fission Yeast Condensin Mutants

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Total RNA (10 μg) was treated with 20 U of DNase I (Promega) at 37 °C for 40 min and then purified by phenol/chloroform extraction. Total RNA (5 μg) was subjected to mRNA purification using oligo d(T)25 beads (NEB #E7490S). cDNA was synthesized from purified RNA (10–40 ng) using NEBNext RNA library prep master mix set for Illumina (NEB #E6110S) and then purified using AMPure XP beads (Beckman Coulter, A63880). cDNA libraries were prepared using NEBNext ultra DNA library prep kit for Illumina (NEB #E7370S) with multiplex oligos (NEB #7500S).
The cDNA samples were sequenced on the Illumina HiSeq 2000 platform to obtain 76-bp single-end reads. Sequenced reads were mapped to the fission yeast reference genome (version ASM294v2.30) using the STAR program (v2.5.2) (ref. 58 (link)). htseq-count software (v0.6.1) was used to estimate read numbers assigned to exons, and the read numbers were normalized using the TMM method^{59 (link),60 (link)}. Differentially expressed genes between wild-type and cut14-208 condensin mutant cells were identified with the edgeR package using the dispersion value of 0.029, which was estimated from the cut3-477 and cut14-208 condensin mutant samples treated as biological replicas^{61 (link)}. FDR < 0.05 was used as a threshold to define significantly up- or downregulated genes.

Tanizawa H., Kim K.D., Iwasaki O, & Noma K.I. (2017). Architectural alterations of the fission yeast genome during the cell cycle. Nature structural & molecular biology, 24(11), 965-976.

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RNA-Seq Library Preparation Protocol

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mRNA was isolated from the total RNA with NEBNext Oligo d(T)25 beads. The fragmentation was performed at 94°C with addition of First Strand Synthesis Reaction Buffer and Random Primer mix (2×). The double-stranded (ds) cDNA was synthetized by using ProtoScript II Reverse Transcriptase and Second Strand Synthesis Enzyme Mix. The blunt ends on the ds cDNA were created with the end-repair reaction. The adapters were ligated using the specific RNA Adapter Indexes supplied in the kit. The PCR reaction was performed to selectively enrich adaptor ligated DNA fragments and create a large amount of DNA, which was then purified using Ampure XP beads. The quantity and quality of libraries were assessed on Rotor-Gene 6000 PCR System (Qiagen, USA) using the qPCR method and on Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip. The final length of the libraries averaged 260 bp. The libraries were normalized to 4 nM, pooled together in equal volumes, and sequenced with 2 × 50 bp paired-end reads on the NextSeq 500 System (Illumina, USA). The obtained data were stored in FASTQ format. The minimum amount of reads for each sample was 20 million.

Moskalev A., Guvatova Z., Shaposhnikov M., Lashmanova E., Proshkina E., Koval L., Zhavoronkov A., Krasnov G, & Kudryavtseva A. (2019). The Neuronal Overexpression of Gclc in Drosophila melanogaster Induces Life Extension With Longevity-Associated Transcriptomic Changes in the Thorax. Frontiers in Genetics, 10, 149.

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PAT-seq Library Preparation for RNA-seq

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Total RNA was extracted with a TaKaRa MiniBEST Plant RNA Extraction Kit and genomic DNA was removed by DNaseI (New England Biolabs). PAT-seq libraries were prepared as previously described with modifications (Lin et al., 2020 (link)). Two micrograms of total RNA were fragmented by 5× first strand buffer (TaKaRa) at 94°C for 4 min. Poly(A) RNAs were then enriched by oligo(dT)₂₅ beads (New England Biolabs). Reverse transcription was performed with oligo d(T)₁₈ primers by SMARTScribe™ Reverse Transcriptase (TaKaRa) for 2 h at 42°C. Then, a modified 5′ adaptor and SMARTScribe Reverse Transcriptase were added for another 2 h at 42°C. The cDNA generated was then purified with AMPure beads and amplified with Phire II (Thermo Fisher Scientific). The amplification products were then separated on a 2% agarose gel and 300–500 bp fragments were purified with a Zymoclean Gel DNA Recovery Kit. The concentration and quality of libraries were tested by a Qubit 2.0 and an Agilent Bioanalyzer 2100, and then sequenced on an Illumina HiSeq 2500 platform with 100-bp rapid sequencing mode.

Ma H., Cai L., Lin J., Zhou K, & Li Q.Q. (2022). Divergence in the Regulation of the Salt Tolerant Response Between Arabidopsis thaliana and Its Halophytic Relative Eutrema salsugineum by mRNA Alternative Polyadenylation. Frontiers in Plant Science, 13, 866054.

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RNA-Seq Library Preparation from HAECs

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HAECs were resuspended in RNA Lysis Buffer and RNA was extracted from cells using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). Selected RNA was fragmented, followed by single strand cDNA synthesis using a SuperScript III First-Strand Synthesis System (ThermoFisher Scientific # 18080051), followed by second strand synthesis using DNA Polymerase I (Qiagen/Enzymatics, Beverly, MA, #P7050L). dsDNA ends were repaired with T4 DNA Polymerase (Enzymatics #P7080L). Barcode adapters (BIOO Scientific NEXTflex, Austin, TX, #514104) were ligated onto the ends of sequences using T4 DNA Ligase (Enzymatics #L-6030-HC-L) and samples were treated with Uracil DNA Glycosylase (UDG) (Enzymatics #G5010L). Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.

Hogan N.T., Whalen M.B., Stolze L.K., Hadeli N.K., Lam M.T., Springstead J.R., Glass C.K, & Romanoski C.E. (2017). Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells. eLife, 6, e22536.

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In Vitro Protein Expression Optimization

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The ligation product was precipitated using co-precipitant, pink (Bioline) and resuspended in 6.5 µL of 10 mM Tris-HCl, pH 7.5. In vitro translation was performed using Solutions A & B of the PURExpress® kit (NEB). Resuspended ligation product was mixed with 1 µL murine RNase inhibitor (NEB), 10 µL Solution A and 7.5 µL Solution B. The reaction was incubated at 37°C for 1.5 hr, followed by incubation on ice for 10 min. 2.5 uL of 1 M MgCl₂ and 12.5 µL of 2 M KCl were added to the reaction, followed by incubation at room temperature for 10 min. The reaction was incubated at −20°C overnight to allow stable fusion formation. Following this step, the fusions were re-purified using the magnetic OligodT₂₅ beads (NEB), as per the manufacturer's instructions and eluted in 30 µL of 10 mM Tris-HCl, pH 7.5. Fusion formation was assayed by western blot using the anti-FLAG-M2 primary antibody (Sigma) to detect the engineered N-terminal FLAG-tag on all the Nbs. The unbound fractions from the oligodT₂₅ purification were used as non-displayed Nb controls (Figure 3d and Figure 5).

Doshi R., Chen B.R., Vibat C.R., Huang N., Lee C.W, & Chang G. (2014). In vitro nanobody discovery for integral membrane protein targets. Scientific Reports, 4, 6760.

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Transcriptomic Analysis of Plant Tissue

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Approximately 100 mg of frozen tissue was then used for total RNA extraction with an OmniPlant RNA Kit based upon provided directions. For RNA-seq analyses, NEBNext, Oligo(dT)25 beads (NEB, USA) were used to specifically enrich for the mRNA present within a 50 μl total RNA sample, after which an NEBNext, Ultra RNA Library Prep Kit for Illumina (NEB) was used to prepare a mRNA library from this enriched samples according to provided directions. An Illumina HiSeq™ 4000 platform was then used for sequencing. The resultant raw reads then underwent quality filtering to remove those reads that were of low quality, contained poly-N sequences, or contained adapter sequences. Clean reads were then de novo assembled with Trinity [51 (link)], thereby producing a transcriptomic reference database.

Cheng Y., Liu H., Tong X., Liu Z., Zhang X., Li D., Jiang X, & Yu X. (2020). Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) seem reveal candidate genes for triterpenoid saponin biosynthesis. BMC Plant Biology, 20, 214.

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Transcriptome Analysis Using RNA-seq

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Approximately 100 mg of frozen tissue was then used for total RNA extraction with an OmniPlant RNA Kit based upon provided directions. For RNA-seq analyses, NEBNext, Oligo(dT)25 beads (NEB, USA) were used to speci cally enrich for the mRNA present within a 50 μl total RNA sample, after which an NEBNext, Ultra RNA Library Prep Kit for Illumina (NEB) was used to prepare a mRNA library from this enriched samples according to provided directions. An Illumina HiSeq TM 4000 platform was then used for sequencing. The resultant raw reads then underwent quality ltering to remove those reads that were of low quality, contained poly-N sequences, or contained adapter sequences. Clean reads were then de novo assembled with Trinity [51] (link), thereby producing a transcriptomic reference database.

Cheng Y., Liu H., Tong X., Liu Z., Zhang X., Li D., Jiang X., & Cheng Y. (2020). Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponin biosynthesis.

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Transcriptome Analysis Using RNA-seq

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Approximately 100 mg of frozen tissue was then used for total RNA extraction with an OmniPlant RNA Kit based upon provided directions. For RNA-seq analyses, NEBNext, Oligo(dT)25 beads (NEB, USA) were used to speci cally enrich for the mRNA present within a 50 μl total RNA sample, after which an NEBNext, Ultra RNA Library Prep Kit for Illumina (NEB) was used to prepare a mRNA library from this enriched samples according to provided directions. An Illumina HiSeq TM 4000 platform was then used for sequencing. The resultant raw reads then underwent quality ltering to remove those reads that were of low quality, contained poly-N sequences, or contained adapter sequences. Clean reads were then de novo assembled with Trinity [51] (link), thereby producing a transcriptomic reference database.

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