dynabeads oligo dt 25, by Thermo Fisher Scientific - Product details

1

RNA-Seq Library Preparation and Sequencing

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Total RNA samples were first treated with DNAse-I to remove any possible DNA contamination, and then the mRNA was enriched using Dynabeads Oligo(dt)₂₅ (Thermo Fisher Scientific, USA). The enriched mRNA was fragmented in smaller fragments of 200 bps approximately, which were attached to adapters with known sequences that were unique for each sample. Samples were connected to magnetic beads containing complementary sequences for the adapters and then inserted in microwells where an emulsion-PCR for cDNA synthesis was carried (illustra Ready-To-Go RT-PCR Beads, GE Lifesciences). Our six cDNA libraries were submitted to quantification and quality control using Agilent 2100 Bioanalyzer and were then loaded in Ion Proton V2 PI chip using the Ion PI™ 200 Sequencing Kit v3 and sequenced using Ion Proton™ (Thermo Fisher Scientific, EUA) platform in a single multiplex run.
Raw data reads obtained by primary sequencing using Ion Proton™ were submitted to quality control to calculate alignment and to assess how the reads behave when compared to the reference human genome (Hg19/GRCh37). The aligned reads were mapped and quantified using TMAP (Torrent Mapping Alignment Program), which supports different alignment algorithms [30 (link)–32 (link)]. Processed datasets were uploaded to GEO (Gene Expression Omnibus) under the access number GSE81265.

Maués J.H., Ribeiro H.F., Pinto G.R., Lopes L.D., Lamarão L.M., Pessoa C.M., Moreira-Nunes C.D., de Carvalho R.M., Assumpção P.P., Rey J.A, & Burbano R.M. (2018). Gastric Cancer Cell Lines Have Different MYC-Regulated Expression Patterns but Share a Common Core of Altered Genes. Canadian Journal of Gastroenterology & Hepatology, 2018, 5804376.

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Purification of mRNA from HEK293T Cells

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HEK293T cells were washed with PBS, then lysed with TRIzol (Thermo Fisher) and cleaned using RNeasy Mini Kit (Qiagen) or Direct-zol RNA Miniprep (Zymo Research) kits with DNaseI treatment following manufacturer’s protocol. Prior to poly(A) enrichment, total RNA was supplemented with 40 nM DNA oligos (Integrated DNA Technologies, Supplementary Table 4) that hybridize to the guide RNA transfected within HEK293T cells and heated at 72 °C for 3 min. Guide-quenching oligos contained a 2’,3’-dideoxycytidine modification at the 3’ end to prevent primer extension in downstream reverse transcription reactions. mRNA was separated from quenched total RNA with Dynabeads Oligo(dT)₂₅ (Thermo Fisher) according to manufacturer’s protocol.

Wilson C., Chen P.J., Miao Z, & Liu D.R. (2020). Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nature biotechnology, 38(12), 1431-1440.

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Methylation-specific RNA Immunoprecipitation

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Total RNA from 4 biological replicates of each condition was poly(A)-enriched using Dynabeads Oligo(dT)₂₅ (Thermo Fisher) and fragmented to a mean size of 200-300 nucleotides by incubation in 50 mM MgCl₂ for 8 min at 95 °C. A portion of fragmented RNA was saved as input. Remaining RNA samples were incubated overnight at 4 °C rotating with protein G magnetic beads (Thermo Fisher) coated with EpiMark anti-m⁶A antibody (New England BioLabs). Washes and elution were performed on a Biomek liquid handler (Beckman Coulter). To remove unbound RNA, samples were washed five times with each of the following buffers: reaction buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), low-salt reaction buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), and high salt reaction buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O). RNA was eluted with RLT buffer (Qiagen) and purified with MyOne Silane Dynabeads (Thermo Fisher). RNA libraries for the RNA input, collected supernatant, and IP were constructed using SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer’s protocol. The size distributions of the resulting libraries were assessed using the Tapestation D1000 screen tape (Agilent Technologies), normalized, and sequenced on a NextSeq 550 (Illumina) using a single read 75 cycle kit.

Wilson C., Chen P.J., Miao Z, & Liu D.R. (2020). Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nature biotechnology, 38(12), 1431-1440.

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Purification of mRNA from P19 Cells

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For library preparation, mRNA from P19 cells before (D0) and during differentiation (D2, D4, and D6) were purified from total RNA using Dynabeads™ Oligo(dT)25 (Thermo Fisher Scientific). Briefly, 20 μL of beads per sample were washed, resuspended in 40 μL of binding buffer (20 mM Tris-HCl pH 7.5, 1.0 M LiCl, and 2 mM EDTA) and mixed with an equivalent volume of water containing 3.5 μg of denatured RNA. After 5 minutes of incubation at room temperature, beads were collected on a magnetic rack and the supernatant was removed. The beads were washed twice in a wash buffer (10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, and 1 mM EDTA) and the mRNA was eluted after a 2-minute incubation at 80°C in 10 μL of Tris-HCl, pH 7.5 for library preparation.

Baptissart M., Papas B.N., Chi R.P., Li Y., Lee D., Puviindran B, & Morgan M. (2023). A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation. iScience, 26(9), 107511.

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Purification of mRNA from HEK293T Cells

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HEK293T cells were washed with PBS, then lysed with TRIzol (Thermo Fisher) and cleaned using RNeasy Mini Kit (Qiagen) or Direct-zol RNA Miniprep (Zymo Research) kits with DNaseI treatment following manufacturer’s protocol. Prior to poly(A) enrichment, total RNA was supplemented with 40 nM DNA oligos (Integrated DNA Technologies, Supplementary Table 4) that hybridize to the guide RNA transfected within HEK293T cells and heated at 72 °C for 3 min. Guide-quenching oligos contained a 2’,3’-dideoxycytidine modification at the 3’ end to prevent primer extension in downstream reverse transcription reactions. mRNA was separated from quenched total RNA with Dynabeads Oligo(dT)₂₅ (Thermo Fisher) according to manufacturer’s protocol.

Wilson C., Chen P.J., Miao Z, & Liu D.R. (2020). Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nature biotechnology, 38(12), 1431-1440.

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Quantifying mRNA m6A Modification

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mRNA was purified from total RNA using Dynabeads® Oligo(dT)25 (Thermo Fisher). Equal amounts of mRNA were spotted to a nylon membrane (Fisher), followed by UV crosslinking at UV 254 nm, 0.12 J/cm². After blocking in PBST containing 5% non-fat milk and 0.1 % Tween-20 for 1 hr, the membrane was incubated with 1:1000 diluted anti-m⁶A antibody overnight at 4 °C. The membrane was incubated with HRP-conjugated anti-rabbit IgG (1:5000 dilution) for 1 hr and visualized by using enhanced chemiluminescence (ECLPlus, GE Healthcare).

Coots R.A., Liu X.M., Mao Y., Dong L., Zhou J., Wan J., Zhang X, & Qian S.B. (2017). m6A Facilitates eIF4F-Independent mRNA Translation. Molecular cell, 68(3), 504-514.e7.

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7

RNA-seq Analysis of Smad and Wnt Signaling

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15*10⁶ COS-7 cells were cultured in DMEM (ThermoFisher Scientific; 31966–021) supplemented with 10% FBS (ThermoFisher Scientific; 10270–106) and 1% Pen/Strep (ThermoFischer Scientific; 15070–063). Cells were transfected with 150 µg library DNA and 75 µg of pcDNA (control), SG4 (15 µg Smad1, 15 µg Smad4, 30 µg Alk3 and 15 µg Gata4 expression vectors) or Wnt (15 µg Tcf4 expression vector, 60 µg pcDNA, and 0.4M LiCl) using Polyethylenimine 25 kDa (PEI; Sigma-Aldrich; 408727) in a ratio of 1:3 (DNA:PEI). Medium was refreshed 6 hr after transfection. 48 hr after transfection, total RNA was isolated using the Qiagen RNeasy maxi prep kit (Qiagen; 75162). polyA+ RNA was isolated using Dynabeads Oligo-dT₂₅ (ThermoFisher Scientific; 61002) and treated with Ambion turboDNase (ThermoFisher Scientifc; AM2238) for 30 min at a maximum concentration of 150 ng/µl. RNA was purified using the Qiagen RNeasy MinElute kit (Qiagen; 74204). First-strand cDNA synthesis and library preparation was performed as described (Arnold et al., 2013 (link)). Library quality and concentration were assessed by Bioanalyzer (Agilent) and KAPA Library Quantification Kit (KAPA Biosystems; KK4824). Libraries were pooled equimolarly and sequenced on an Illumina MiSeq (PE150).

van Weerd J.H., Mohan R.A., van Duijvenboden K., Hooijkaas I.B., Wakker V., Boukens B.J., Barnett P, & Christoffels V.M. (2020). Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction. eLife, 9, e56697.

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8

Methylation-specific RNA Immunoprecipitation

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Total RNA from 4 biological replicates of each condition was poly(A)-enriched using Dynabeads Oligo(dT)₂₅ (Thermo Fisher) and fragmented to a mean size of 200-300 nucleotides by incubation in 50 mM MgCl₂ for 8 min at 95 °C. A portion of fragmented RNA was saved as input. Remaining RNA samples were incubated overnight at 4 °C rotating with protein G magnetic beads (Thermo Fisher) coated with EpiMark anti-m⁶A antibody (New England BioLabs). Washes and elution were performed on a Biomek liquid handler (Beckman Coulter). To remove unbound RNA, samples were washed five times with each of the following buffers: reaction buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), low-salt reaction buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), and high salt reaction buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O). RNA was eluted with RLT buffer (Qiagen) and purified with MyOne Silane Dynabeads (Thermo Fisher). RNA libraries for the RNA input, collected supernatant, and IP were constructed using SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer’s protocol. The size distributions of the resulting libraries were assessed using the Tapestation D1000 screen tape (Agilent Technologies), normalized, and sequenced on a NextSeq 550 (Illumina) using a single read 75 cycle kit.

Wilson C., Chen P.J., Miao Z, & Liu D.R. (2020). Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nature biotechnology, 38(12), 1431-1440.

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9

mRNA Isolation from Cultured Cells

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Twenty-four hours after electroporation, HEK293/HeLa cells were lysed using TRIzol (Thermo Fisher Scientific, Cat# 15596026). Each 500 μl TRIzol-lysed solution was mixed with 100 μl chloroform (Fisher Chemical, Cat# C298-500) to allow phase separation. The upper aqueous phase was transferred and mixed with equal volume ethanol (200 proof, Fisher BioReagents). The mixture was loaded to the column supplied by the Direct-zol RNA Miniprep Plus kit (Zymo Research, Cat# R2072) to isolate total RNA following the manufacturer’s protocol. PolyA selection was carried out to isolate mRNA from total RNA using Dynabeads™ Oligo(dT)₂₅ (Thermo Fisher Scientific, Cat# 61002). The concentration of mRNA in each sample was quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat# Q32852) with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific).

Fu T., Amoah K., Chan T.W., Bahn J.H., Lee J.H., Terrazas S., Chong R., Kosuri S, & Xiao X. (2024). Massively parallel screen uncovers many rare 3′ UTR variants regulating mRNA abundance of cancer driver genes. Nature Communications, 15, 3335.

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mRNA Isolation and Illumina Library Preparation

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Total RNA and Dynabeads® Oligo (dT) 25 (Thermo Fisher Scientific, Waltham, MA, USA) were used to isolate mRNA. The resulting mRNA fragments of ~400 nucleotides were converted to double-stranded complementary DNA (cDNA) using random hexamer primers and corresponding enzymes in accordance with standard DNA library protocols for sequencing. Briefly, cDNA was end-repaired, phosphorylated, and adenylated. Common TruSeq adapters containing 8-bp indexes (i5 and i7) suitable for Illumina sequencing were then ligated to the adenylated molecules, and the resulting libraries were amplified by 13 cycles of PCR to enrich for properly ligated molecules (Figure 1b). The final libraries were quantified using PicoGreen (Thermo Fisher Scientific) and equally combined into a single sample, which was then sequenced on an Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) instrument. Paired-end reads with an average length of 100 bp were obtained. Library preparation and sequencing were carried out by RAPiD Genomics, LLC (Gainesville, FL, USA).

Torres-Silva G., Correia L.N., Batista D.S., Koehler A.D., Resende S.V., Romanel E., Cassol D., Almeida A.M., Strickler S.R., Specht C.D, & Otoni W.C. (2021). Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction. Frontiers in Plant Science, 12, 697556.

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Dynabeads oligo dt 25

Lab products found in correlation

174 protocols using dynabeads oligo dt 25

RNA-Seq Library Preparation and Sequencing

Purification of mRNA from HEK293T Cells

Methylation-specific RNA Immunoprecipitation

Purification of mRNA from P19 Cells

Purification of mRNA from HEK293T Cells

Quantifying mRNA m6A Modification

RNA-seq Analysis of Smad and Wnt Signaling

Methylation-specific RNA Immunoprecipitation

mRNA Isolation from Cultured Cells

mRNA Isolation and Illumina Library Preparation

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