Ribosome profiling was carried out as described previously (Li et al., 2012 (link); Oh et al., 2011 (link)) with the following modifications. To accurately capture the ribosome occupancy on mRNAs with single-codon resolution, we flash-froze the cells immediately upon harvesting and stabilized ribosomes with the translation inhibitor chloramphenicol only at the lysis stage. Cells were lysed using glass beads (G1277, Sigma, vortex 10 × 30 s at 4°C with 60 s cooling on ice in between). Micrococcal nuclease digestion was carried out with 1 U Worthington Biochemicals MNase per μg of nucleic acid as measured by A260. Monosome-protected mRNA footprints between 20 nt and 40 nt were size-selected by polyacrylamide gel electrophoresis for monosome sequencing. For disome sequencing, the disome peak was collected from the MNase-treated polysomes after sucrose-gradient fractionation, and fragments of size between 50 nt and 80 nt were used for sequencing. For total mRNA sequencing, the Microbe Express kit (Ambion) was used for subtracting rRNA from total mRNA and then fragmented using a bicarbonate buffer (Ingolia et al., 2009 (link)) for 20 min. For library construction, polyA-tailing (Ingolia et al., 2009 (link)) was used instead of linker ligation.
G1277
Manufactured by Merck Group
The G1277 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, providing a reliable and consistent solution for various applications. The core function of the G1277 is to perform specific tasks required in a laboratory environment. Detailed technical specifications and intended use are not provided in this unbiased and factual description.
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Lab products found in correlation
Microbe express kit, by Thermo Fisher Scientific (1 mentions)
G4649, by Merck Group (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Quick rna miniprep, by Zymo Research (1 mentions)
4 protocols using g1277
1
Ribosome Profiling: Detailed Protocol
2
Protein Extraction from A. brasilense
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A. brasilense strains were grown in NB media at 30°C with shaking and bacteria in logarithmic phase (A600 0.5–0.8) were collected by centrifugation. For flocculation conditions, bacteria were collected by centrifugation after 3–4 hours shaking at 30°C in flocculation medium [6] (link). About 50 mg cells were lysed and homogenized using a mini beadbeater containing a mixture of glass beads (G4649 and G1277, Sigma) and lysis buffer (30% sucrose, 0.1 M Tris pH 8.0, 2 mM PMSF, 1% DTT, 100 mM KCl, 5 mM EDTA) 4 times for 30 sec. Protein extraction was performed from the lysates by phenol extraction [16] (link) and proteins were precipitated overnight using 5 volumes of 0.1 M ammonium acetate in methanol at −20°C. The protein solution was subsequently centrifuged at 6,000 g for 10 min and the pellet rinsed twice with 0.1 M ammonium acetate in methanol, three times with cold methanol and once with 80% acetone. The protein pellet was then cold-dried using a vacuum freeze dryer for 4 hours, then dissolved in IEF buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 0.5% IPG buffer pH 3–10, 1% DTT, 0.2% Coomassie Brilliant Blue). The protein concentration was determined using the 2D Quant Kit (GE Healthcare Life Science, Australia).
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A. brasilense strains were grown in NB media at 30°C with shaking and bacteria in logarithmic phase (A600 0.5–0.8) were collected by centrifugation. For flocculation conditions, bacteria were collected by centrifugation after 3–4 hours shaking at 30°C in flocculation medium [6] (link). About 50 mg cells were lysed and homogenized using a mini beadbeater containing a mixture of glass beads (G4649 and G1277, Sigma) and lysis buffer (30% sucrose, 0.1 M Tris pH 8.0, 2 mM PMSF, 1% DTT, 100 mM KCl, 5 mM EDTA) 4 times for 30 sec. Protein extraction was performed from the lysates by phenol extraction [16] (link) and proteins were precipitated overnight using 5 volumes of 0.1 M ammonium acetate in methanol at −20°C. The protein solution was subsequently centrifuged at 6,000 g for 10 min and the pellet rinsed twice with 0.1 M ammonium acetate in methanol, three times with cold methanol and once with 80% acetone. The protein pellet was then cold-dried using a vacuum freeze dryer for 4 hours, then dissolved in IEF buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 0.5% IPG buffer pH 3–10, 1% DTT, 0.2% Coomassie Brilliant Blue). The protein concentration was determined using the 2D Quant Kit (GE Healthcare Life Science, Australia).
3
Total RNA Isolation from Bacterial Cells
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Total RNA was isolated from cells grown aerobically until late stationary phase (18 h growth) in LB at 37°C, using TRIzol. Pellets of cells were resuspended in 12.5 mM Tris-HCl (pH 7.6), 10 mM EDTA, 10% glucose. After addition of 1/5 volume of 0.5 M EDTA, disruption of cells was performed by vigorous shaking using glass beads (G1277, Sigma-Aldrich) in acid phenol pH 4.5 (Interchim). After centrifugation, the aqueous phase was carefully mixed with 2 volumes of TRIzol (Invitrogen), and five minutes later with a chloroform-isoamyl alcohol mixture (24∶1). After centrifugation, chloroform-isoamyl alcohol was added to the aqueous phase and the mixture was allowed to stand for five minutes before centrifugation. Total RNA present in the aqueous phase was precipitated with isopropanol. After centrifugation, the pellet was washed in 70% Ethanol, air-dried and resuspended in RNAse-free water. The RNA was subsequently treated with DNaseI (Ambion) and its quality was analyzed using an Agilent BioAnalyzer.
4
Phenol-Chloroform RNA Extraction Protocol
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Phenol-chloroform extraction method was used to obtain total RNA. 10 ml of cells were quickly chilled in an ice water bath and harvested by centrifugation at 3000 g for 5 min. Cell pellets were re-suspended in 500 µl of 0.3 M sodium acetate and 10 mM EDTA pH 4.5. Re-suspended cells were mixed with 500 µl of acetate-saturated phenol-chloroform pH 4.5 and 500 µl of acid-washed glass beads (G1277, Sigma). The mixture was shaken in a vortexer for 3 min and then clarified by centrifugation at 21,000 g for 10 min. The samples were maintained at 4°C through this step. The aqueous layer was extracted twice with acetate-saturated phenol-chloroform pH 4.5 and once with chloroform. Total RNA was precipitated with an equal volume of isopropanol, washed with 70% ethanol, and finally re-suspended in 200 µl of RNase-free 10 mM Tris pH 7.0. 200 ng of the total RNA was treated with DNase I (M0303S, NEB) to remove residual DNA contamination (manufacturer’s instructions were followed). The DNA-free RNA was column-purified (Quick-RNA Miniprep, Zymo Research, Irvine, CA).
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