A total of 2000 O.D.260 units of MBP was digested by 3000 units of RNase I (Ambion) in 400 µl digestion buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 5 mM MgCl2, 50 µg/ml cycloheximide, 50 µg/ml chloramphenicol) with gentle shaking at room temperature for 1.5 hr, and the digestion product was centrifuged in a 15–50% sucrose density gradient at 300,000 g for 1.5 hr. A gradient fractionation system (ISCO) was used to collect the monosome fraction. Monosomal RNAs were extracted with TRI Reagent (MRC), and resolved on 15% Urea-PAGE. RNAs corresponding to 25–35 nt were recovered from the gel and phosphorylated by T4 polynucleotide kinase (NEB). Next, the monosomal RNAs were further polyadenylated with PolyA Polymerase (NEB), and reverse transcribed with the primer oligodT linkerM (Supplementary file 5 ), followed by cDNA circularization with CircLigase (EpiCenter). rRNAs were removed by hybridization with biotin conjugated DNA probes (Supplementary file 5 ) followed by capture with Dynabead M-280 Streptavidin (Invitrogen). Finally, the Ribo-seq library was obtained by PCR with primers listed in Supplementary file 5 .
Gradient fractionation system
Manufactured by Teledyne
The Gradient fractionation system is a laboratory equipment designed to separate and purify complex mixtures of biomolecules, such as proteins, nucleic acids, or cellular organelles. The system utilizes a density gradient, created by a continuously varying concentration of a solute, to facilitate the separation of different components based on their density differences. The core function of this system is to enable efficient and reproducible fractionation of samples, which is a crucial step in many biological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Igepal ca 630, by Merck Group (2 mentions)
Sw 32.1 ti rotor, by Beckman Coulter (2 mentions)
Recombinant rnasin, by Promega (2 mentions)
Circligase, by Illumina (1 mentions)
Dynabead m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Poly a polymerase, by New England Biolabs (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Amplex red glucose glucose oxidase assay kit, by Thermo Fisher Scientific (1 mentions)
Lactate assay kit, by Abcam (1 mentions)
4 protocols using gradient fractionation system
1
Ribosome Profiling via Ribo-Seq
2
CD4+ T cell Polysome Profiling
3
CD4+ T cell Polysome Profiling
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CD4+ T cells activated under nonpolarizing conditions with plate-bound anti-CD3 and anti-CD28 were harvested on day 4 post-activation. Cycloheximide (0.1 mg/ml) was added to the cell cultures 15 min before the cells were collected. Cells were pelleted and washed three times with ice-cold PBS containing 0.1 mg/ml cycloheximide. Cytoplasmic extracts were carefully layered over 10–50% linear sucrose gradients in polysome buffer (10 mM HEPES [pH 7.5], 100 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 50 U recombinant RNasin [Promega], and 0.1% IGEPAL CA-630 [Sigma-Aldrich]) and centrifuged at 17,000 rpm in a Beckman SW 32.1 Ti Rotor for 4 h at 4°C. Gradients were fractioned using an Isco gradient fractionation system equipped with a UA-6 detector. Light RNP fractions 40S, 60S, and 80S and heavy polysome fractions were monitored by the continuous UV-absorption profile at A254. Nine fractions were collected, and RNAs associated with each fraction were isolated using TRIzol extraction. RNAs from each fraction were reverse transcribed followed by qPCR. The percentage distribution of RNA in the 40S, 60S, 80S, and heavy polysome fractions was analyzed.
4
Glucose Uptake and Lactate Production Assay
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The Amplex® Red Glucose/Glucose Oxidase Assay Kit (Invitrogen, Cat.#A22189) was used to measure the glucose uptake. Glucose consumption was calculated by the net content of the original glucose concentration deduced the measured glucose concentration in the medium. The Lactate Assay Kit (BioVision, Cat.#ABIN411683) was used to measure lactate production. Total proteins were used for the normalization of the results obtained above. These experiments were performed in triplicate and repeated twice.
CCK8 assay 1×10 3 cells were seeded into 96-well plates. Adding detection reagent (10μl CCK8 reagent+ 90μl DMEM) to each plate and incubating at 37℃for 1h. Measure the absorbance at 450nm with a microplate reader.
Each experiment was carried out independently in triplicate.
Polysome fractionation and RNA isolation cellular extracts were centrifuged at 10,000 rpm for 5 min at 4°C and the supernatant was carefully isolated and loaded onto 10-50% sucrose gradients containing 0.1 mg/ml heparin and 2mM DTT and centrifuged at 37,000 rpm for 2.5 h at 4°C (SW40 rotor). The sucrose gradient was subsequently fractionated with a gradient fractionation system (ISCO) connected to a UV detector to monitoring absorbance at 252nm. RNA was isolated from polysomal fractions using the PureLink RNA Mini Kit (Invitrogen).
CCK8 assay 1×10 3 cells were seeded into 96-well plates. Adding detection reagent (10μl CCK8 reagent+ 90μl DMEM) to each plate and incubating at 37℃for 1h. Measure the absorbance at 450nm with a microplate reader.
Each experiment was carried out independently in triplicate.
Polysome fractionation and RNA isolation cellular extracts were centrifuged at 10,000 rpm for 5 min at 4°C and the supernatant was carefully isolated and loaded onto 10-50% sucrose gradients containing 0.1 mg/ml heparin and 2mM DTT and centrifuged at 37,000 rpm for 2.5 h at 4°C (SW40 rotor). The sucrose gradient was subsequently fractionated with a gradient fractionation system (ISCO) connected to a UV detector to monitoring absorbance at 252nm. RNA was isolated from polysomal fractions using the PureLink RNA Mini Kit (Invitrogen).
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