Artseq ribosome profiling kit

Manufactured by Illumina

The ARTseq Ribosome Profiling Kit is a laboratory instrument designed for the analysis of ribosome-protected mRNA fragments, enabling the study of translation dynamics and gene expression regulation. The kit provides a standardized workflow for the isolation, preparation, and sequencing of ribosome-protected mRNA fragments.

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Lab products found in correlation

Hiseq 2000, by Illumina (4 mentions) Hiseq 2500, by Illumina (3 mentions) Fragment analyzer, by Agilent Technologies (2 mentions) Ribo zero gold kit, by Illumina (2 mentions) Superase in, by Thermo Fisher Scientific (2 mentions) Truseq stranded mrna sample prep kit, by Illumina (2 mentions) Htseq2000, by Illumina (2 mentions) Rnase 1, by Thermo Fisher Scientific (2 mentions) 0.45 μm filter, by Merck Group (1 mentions) Hiseq 2000 platform, by Illumina (1 mentions) Dynabeads mrna direct micro purification kit, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by MedChemExpress (1 mentions) Optima l 80 ultracentrifuge, by Beckman Coulter (1 mentions) Biologic lp chromatography system, by Bio-Rad (1 mentions) Kapa library quantification kit, by Roche (1 mentions) Hiseq 2500 device, by Illumina (1 mentions) Trizol ls, by Thermo Fisher Scientific (1 mentions) C7698, by Merck Group (1 mentions) Ribo zero gold, by Illumina (1 mentions) Ribo zero magnetic kit, by Illumina (1 mentions)

19 protocols using artseq ribosome profiling kit

Ribosome Profiling and RNA-seq for Translational Analysis

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Ribosomal profiling (Ribo-seq) was performed using the ARTseq-TM Ribosome Profiling kit from Illumina (#RPHMR12126, Illumina) according to the manufacturer's instructions [30 (link)]. For final amplification of the library, 12 PCR cycles were run with subsequent library purification. The quality and quantity of the samples were determined using a Fragment Analyzer (Advanced Analytical Technologies).
Reads were mapped against the human genome (hg38) using STAR [69 (link)] and processed based on annotations from Ensembl (v86). A generative model was fitted to the observed positions of annotated codons in strongly translated ORFs. Translated ORFs were determined and each ORF was quantified using the number of reads mapped to its codons.
For corresponding RNA-sequencing analysis, reads were mapped against the human genome (hg38) using STAR. Then, reads per gene were counted when they were consistent with at least one transcript annotated in Ensembl v86.

Schmidt S., Gay D., Uthe F.W., Denk S., Paauwe M., Matthes N., Diefenbacher M.E., Bryson S., Warrander F.C., Erhard F., Ade C.P., Baluapuri A., Walz S., Jackstadt R., Ford C., Vlachogiannis G., Valeri N., Otto C., Schülein-Völk C., Maurus K., Schmitz W., Knight J.R., Wolf E., Strathdee D., Schulze A., Germer C.T., Rosenwald A., Sansom O.J., Eilers M, & Wiegering A. (2019). A MYC/GCN2/eIF2α negative feedback loop limits protein synthesis to prevent MYC-dependent apoptosis in colorectal cancer. Nature cell biology, 21(11), 1413-1424.

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Ribosome Profiling and RNA-seq for Translational Analysis

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Yeast Ribosome Profiling Protocol

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Yeast cultures were inoculated into 500 ml of standard YPD medium at the initial OD₆₀₀ < 0.1 and incubated at 30°C with shaking until the OD₆₀₀ reached 0.5. Cells were collected by filtering through 0.45 μm filter (Millipore) with glass holder. Pellets were scraped with spatula, flash frozen in liquid nitrogen and stored at −80°C. Yeast extracts were prepared by cryogrinding the cell paste with BioSpec cryomill. Aliquots of cell lysates were used for extraction of footprints (short ribosome-protected mRNA fragments) and isolation of total RNA. The RNA-seq and Ribo-seq libraries were prepared using ARTseq Ribosome Profiling kit (Illumina) as described previously (Labunskyy et al., 2014 (link)) and sequenced using the Illumina HiSeq 2000 platform.

Beaupere C., Wasko B.M., Lorusso J., Kennedy B.K., Kaeberlein M, & Labunskyy V.M. (2017). CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes. Cell reports, 18(8), 1884-1892.

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Ribosome Profiling of Msi2 Knockdown

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Ribosome profiling was performed on scrambled shRNA and Msi2 knockdown keratinocytes using the ART-Seq Ribosome profiling kit (Illumina). Briefly, cultured cells were grown to ∼80% confluency on a 15 cm plate and treated with 50 ug/ml cyclohexamaide for 1 min before they were lysed, aliquoted and digested with RNase. Ribosomes and associated RNA were isolated using illustra™ MicroSpin™ S-400 HR Columns (Illumina). RNA was extracted from the isolate and rRNA was depleted once using Ribo-Zero Gold™ kit (Illumina). RNA fragments 28–32 nts long were isolated via denaturing PAGE gel, ligated to a 3′ adapter, reverse transcribed, circularized using CircLigase, depleted for rRNA a second time and PCR amplified following the provided protocol. RNA-seq was performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) with minor modifications using lysate matched to the ribosome profiling samples. Briefly, mRNA was isolated from total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher) and fragmented for 15 min at 94°C. First strand synthesis, second strand synthesis, end repair, adapter ligation and PCR were performed as described in provided protocol. All PCR products were sequenced on an Illumina HiSeq 2000 using 1 × 100 sequencing.

Bennett C.G., Riemondy K., Chapnick D.A., Bunker E., Liu X., Kuersten S, & Yi R. (2016). Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration. Nucleic Acids Research, 44(8), 3788-3800.

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Polysome Profiling of Fungal Strains

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The culture condition for polysome profiling is the same as the metabolic labeling experiment. Cultures of the WT and Δcpc-3 strains were frozen in liquid nitrogen immediately after collection. Tissue samples were grounded into powder in liquid nitrogen and equal volume of the tissue powder of each sample was added the same volume of lysis buffer (1 × polysome buffer in ARTseq Ribosome Profiling Kit (Illumina, Cat. No. RPYSC12116), 1% Triton X-100, 0.1 mg/ml CHX, 1× protease inhibitor cocktail (EDTA-free, MedChemExpress, Cat. No. HY-K0010), 0.2 U/μl SUPERase•In (Invitrogen, Cat. No. AM2694) and 2 mM DTT). The lysates were then centrifugated at 15 000 rpm for 10 min before the A₂₆₀ of the supernatant was measured by NanoDrop Microvolume Spectrophotometer. The A₂₆₀/ml of the lysate was calculated according to the protocol for the ARTseq Ribosome Profiling Kit. The same OD amount (20 OD_260nm) of the lysate for each sample was loaded onto 10–50% sucrose gradient buffer containing 20 mM HEPES (pH 7.6), 0.1 M KCl, 5 mM MgCl₂, 10 μg/ml CHX, the 1× protease inhibitor cocktail and 10 units/ml SUPERase•In. The sucrose gradients were then centrifuged at 35 000 rpm for 2 h at 4°C using a SW41Ti rotor in a Beckman Coulter (Optima L-80 ultra) centrifuge. Sucrose gradients were analyzed using a BioLogic LP chromatography System (Bio-Rad, Cat. No. 731-8350).

Lyu X., Yang Q., Zhao F, & Liu Y. (2021). Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed. Nucleic Acids Research, 49(16), 9404-9423.

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Transcriptomic and Translational Profiling of B Cells

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mRNAseq libraries were obtained using TruSeq Stranded mRNA Sample Prep Kit (Illumina Inc). Splenic B cells from individual HuR^f/f control or HuR-cKO mice were independently processed for RNA extraction (ex vivo samples, n=4 per genotype) or were stimulated with LPS for 48 hours in RPMI media plus 1 mM NaPyr (n=3-4 per genotype).
Previously published mRNAseq libraries were used to analyse the expression of genes involved in glycolysis, TCA cycle and electron transport in naive and GC B cells (GEO deposition number - GSE47705)^{54 (link)}.
Ribosome footprinting profiling (Ribo-Seq) assays were performed using ARTseq^™ Ribosome Profiling Kit (Epicentre, Illumina). Ex vivo or LPS-activated B cells (n=4-5 per genotype and condition) were treated with cyclohexamide (CHX, 100 μg/ml) three minutes prior cell extract preparation. cDNA libraries were sequenced using either GAIIX (iCLIP) or HTSeq2000 (mRNAseq and Ribo-Seq) Illumina technology. The type of sequencing performed was: iCLIP, 40 bp SE; mRNAseq, 100 bp SE; and Ribo-seq, 50 bp SE.

Diaz-Muñoz M.D., Bell S.E., Fairfax K., Monzon-Casanova E., Cunningham A.F., Gonzalez-Porta M., Andrews S.R., Bunik V.I., Zarnack K., Curk T., Heggermont W.A., Heymans S., Gibson G.E., Kontoyiannis D.L., Ule J, & Turner M. (2015). The RNA-binding protein HuR (Elavl1) is essential for the B cell antibody response. Nature immunology, 16(4), 415-425.

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Ribosome profiling of macrophage transcriptome

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Genome-wide assessment of ribosome-occupancy was assessed using ARTSeq Ribosome profiling kit (Epicentre). Briefly, 10 × 10⁶ macrophages were seeded on a 15 cm plate the day before induction of TTP expression with doxycyclin (6 h treatment) and stimulation with LPS for 1 h. Cells were rinsed twice with ice cold 1× PBS containing cycloheximide (0.1 mg/ml) before cell extract preparation (≈1 ml). One tenth (100 μl) of the sample was used for total RNA isolation and library preparation using the Ribo-Zero Gold Kit (Epicentre) and following instructions in the ARTSeq Ribosome profiling kit. 200 μl of the samples were used for nuclease digestion and ribosome-protected RNA purification prior to cDNA library preparation. cDNA library size was assessed using High Sensitivity DNA Analysis BioAnalyzer kits (Agilent) and libraries were quantified using KAPA Library Quantification Kits (Kapa Biosystems). 50 bp SE multiplexed sequencing was done on an Illumina HiSeq2500 device.

Tiedje C., Diaz-Muñoz M.D., Trulley P., Ahlfors H., Laaß K., Blackshear P.J., Turner M, & Gaestel M. (2016). The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation. Nucleic Acids Research, 44(15), 7418-7440.

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Ribosome Profiling Using ARTseq Kit

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Ribosome profiling experiments were performed using the ARTseq Ribosome profiling kit (Epicentre RPHMR12126), with RNA extraction by Trizol LS (Ambion), rRNA depletion via RiboZero Gold (Epicentre MRZG126), and quality and quantity of small RNA and DNA assayed using Agilent High Sensitivity Small RNA kit and DNA kit, respectively (Agilent 5067-1548, 5067-4626). Sequencing was performed at the UCSF sequencing core on Illumina HiSeq2000, and analysis of reads was performed using Babel^{20 (link)}. Cycloheximide was made fresh to 50 mg/ml in ethanol for each experiment, used at a concentration of 100 μg/ml (Sigma-Aldrich C7698).

Goldsmith J., Marsh T., Asthana S., Leidal A.M., Suresh D., Olshen A, & Debnath J. (2020). Ribosome profiling reveals a functional role for autophagy in mRNA translational control. Communications Biology, 3, 388.

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Optimized Ribosome Profiling of Kidney and Liver

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Generation of liver RPF-seq and RNA-seq libraries using the ARTseq ribosome profiling kit (Epicentre) was described recently [10 (link), 16 (link)]. Kidney libraries were prepared in the same manner, with a single modification to the order of steps in RPF library preparation. After RNase treatment and recovery of ribosome-protected fragments, 5 μg of material was first ribosomal RNA (rRNA)-depleted (Ribo-Zero magnetic kit, Epicentre) and then purified by 15% PAGE. In the formerly prepared liver libraries, Ribo-Zero treatment and PAGE purification had been inverted because at the time we had found that changing the order had a beneficial effect on obtaining highly concentrated libraries. For the kidney samples, however, we noted that this modified order led to higher contamination with reverse-strand rRNA probes bleeding from the Ribo-Zero kit and we thus reverted to ARTseq’s original order. All other steps and materials were identical between liver and kidney samples and followed the ARTseq ribosome profiling kit instructions. RPF and RNA libraries were sequenced on an Illumina HiSeq 2500.

Castelo-Szekely V., Arpat A.B., Janich P, & Gatfield D. (2017). Translational contributions to tissue specificity in rhythmic and constitutive gene expression. Genome Biology, 18, 116.

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Ribosome Profiling of Canine Transcriptome

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Next-generation sequencing libraries of ribosome-protected fragments and total RNA were prepared using the ARTseq Ribosome Profiling Kit (epicentre) according to the manufactures protocol with the following modification. After RNase I treatment of rough microsomes 80S monosomes were isolated by sucrose gradient and protected mRNA fragments were recovered by dissociation of the monosomes into subunits. Raw single-end sequencing reads were clipped of the known adaptor sequence. Clipped reads were first mapped to Canis lupus familiaris rRNA, tRNA and mitochondrial non-coding RNA using Bowtie 1.0.0. Unaligned reads were then mapped to the dog genome assembly (CanFam3.1, Ensemble release 77) using GSNAP (version 2013-10-10) including annotated splice junctions. Unique aligned reads on protein coding genes were counted with HTSeq 0.6.1 and normalized for the length of the coding sequence. For total mRNA sequencing RNase I digestion was omitted.

Pfeffer S., Burbaum L., Unverdorben P., Pech M., Chen Y., Zimmermann R., Beckmann R, & Förster F. (2015). Structure of the native Sec61 protein-conducting channel. Nature Communications, 6, 8403.

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