Approximately 5 × 107 P19 cells were irradiated with 0.25 J/cm2 UV light at 254 nm, harvested, and lysed as previously described (Castello et al. 2012 (link)). Poly(A)+ mRNAs and cross-linked proteins were captured with oligo(dT)25 magnetic beads (New England Biolabs). Oligo(dT)25 beads were washed with buffers containing decreasing concentrations of LiCl and LiDS, and cross-linked proteins were eluted for 3 min at 55°C , concentrated, and loaded on a 4%–12% NuPAGE gel (Life Technologies). Released NXF1 was analyzed by Western blotting using NXF1-specific antibodies (Santa Cruz Biotechnology).
Oligo dt 25 beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Oligo(dT)25 beads are a type of magnetic beads coated with oligo(dT) sequences. They are used for the isolation and purification of polyadenylated (poly(A)) RNA from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Oligo d t 25 magnetic beads, by New England Biolabs (1 mentions)
Q5 master mix, by New England Biolabs (1 mentions)
Nextseq, by Illumina (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
T4 rna ligase 1, by New England Biolabs (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
Circligase 2, by Illumina (1 mentions)
Agencourt ampure xp, by Beckman Coulter (1 mentions)
Rnaprep pure plant kit polysaccharides and polyphenolics rich, by Tiangen Biotech (1 mentions)
6 protocols using oligo dt 25 beads
1
Isolation and Analysis of UV-Induced RNA-Binding Proteins
2
RNA-Seq Library Preparation Protocol
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RNA-Seq libraries were prepared as described in [45 (link)]. Briefly, 10 μg of total RNA was polyA selected twice using Oligo(dT)25 beads (Life Technologies) and NEB oligo(dT) binding buffer. PolyA-selected RNA was fragmented, repaired, and cleaned using Zymo RNA concentrator-5 kit. A total of 30 ng of polyA-selected RNA per sample were used to make RNA-Seq libraries. An adapter was ligated to RNA, RNA was reverse transcribed, and a second adapter was ligated on cDNA. Illumina indexes were introduced during nine cycles of PCR using NEB Q5 Master Mix. Samples were sequenced 100-index-100 on HiSeq2500.
3
Adapted PSI-seq Method for Ribosome Profiling
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The PSI-seq method was adapted from Lovejoy et al. (2014) (link) with changes in initial RNA isolation, CMCT removal, and the sizes of RNA, cDNA, and DNA extracted from gels (Ingolia et al. 2012 (link)). Specifically, total RNA was isolated from heavily infected HFFs using phenol chloroform extraction, and mRNA was isolated using Oligo(dT)25 beads (Thermo Fisher). RNA was then treated as detailed in Lovejoy et al. (2014) (link), with the exception that CMCT removal was performed with buffer at pH 11.4 rather than 10.4 (as detailed in Primer Extension). Additionally, RNAs extracted from the gel were split into fragments 100–200 (“small subset”) and 200–300 (“large subset”) nucleotides long; these subsets were kept separate through linker ligation and cDNA isolation, and were recombined before library circularization. Amplified libraries were submitted for sequencing on Illumina GAII and NextSeq platforms as 1 × 50 bp reads.
4
Mapping Exonucleolytic Cleavage Sites in MYL9 Transcript
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Mapping of exonucleolytic cleavage site in the MYL9 transcript was performed essentially as described previously.4 In brief, PolyA+ RNA was extracted from purified CD34+ cord blood HSCs using Oligo(dT)25 Dynabeads (Thermo Fisher Scientific). One µg of the purified RNA was then incubated with a 5′‐CACGACGCUCUUCCGAUCU RNA oligonucleotide adaptor and T4 RNA ligase 1 (New England Biolabs, Ipswich, USA). This results in a ligation of the adaptor to any RNAs with a 5′ phosphate, a hallmark of endonucleolytic cleavage. The reaction was then repurified on the Oligo(dT)25 beads to remove excess adaptor and reverse transcribed with random hexamers and Superscript III (Thermo Fisher Scientific). Tiled PCRs were then performed using the 5′ adaptor primer in combination with the reverse primers MYL9‐213, MYL9‐257, MYL9 372 or MYL9r, which are spaced at regular interval in the MYL9 mRNA (Supplementary table 2 ). The resulting PCR products were Sanger sequenced to determine the adaptor‐MYL9 ligation point and therefore the original cleavage site.
5
2P-seq of nuclear RNA with modifications
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2P-seq was performed as described before (Spies et al., 2013 (link)) with several modifications. First, nuclear RNA was used instead of total RNA. Second, the primer sequence was re-designed (Supplementary file 2 ), so that multiple libraries could be sequenced in the same lane using Illumina sequencer. Third, a lower concentration of RT primer was used during reverse transcription to reduce internal priming (Scotto-Lavino et al., 2006 (link)). Briefly, poly(A) RNA was purified using oligo-dT25 beads (Invitrogen) and eluted directly into 25 μl of RNase T1 buffer. RNase T1 digestion was performed for 20 min at 22°C using 0.5 U RNase T1 (biochemistry grade, Ambion). After this partial RNase T1 digestion, reverse transcription was performed by addition of 1 μl 1 μM RT primer. Single-stranded cDNAs of 200–400 nucleotides were purified on a 6% TBE-urea gel, then circularized by CircLigaseII (Epicentre). Circularized cDNA was amplified by high-fidelity PCR for 10 cycles using barcoded PCR primers, then gel purified and sequenced using the Illumina Read one primer.
6
RNA-Seq Library Preparation from Plant Tissues
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Total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides and Polyphenolics-rich) (Tiangen Biotech, Beijing, China). For RNA-Seq library construction, 5 μg of total RNA with an appropriate amount of Oligo-dT25 beads (Invitrogen, Carlsbad, CA, USA) was used. The mRNA was fragmented into short fragments and reverse transcribed into cDNA by random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H and dNTPs. After that step, the cDNA fragments were purified by Agencourt AMPure XP (Beckman Coulter, Pasadena, CA, USA). The purified cDNA fragments were end-repaired, modified with poly (A) tails, and ligated to Illumina sequencing adapters. The size-selected fragments were amplified and purified. Sixteen libraries were sequenced withIllumina HiSeq™ 2500 (BGI Co. Ltd., Shenzhen, China). The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive [12 (link)] in the Beijing Institute of Genomics (BIG) Data Center [13 (link)], Beijing Institute of Genomics, Chinese Academy of Sciences, under accession numbers CRA001770 and are publicly accessible at https://bigd.big.ac.cn/gsa .
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