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| Status |
Public on Jan 30, 2017 |
| Title |
Ribosome profiling of wild type Salmonella enterica serovar Thyphimurium - strain SL1344 polysomes |
| Sample type |
SRA |
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| Source name |
Salmonella enterica serovar Thyphimurium - strain SL1344
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
strain: SL1344
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| Treatment protocol |
Bacterial cells were pre-treated for 5 min with chloramphenicol (Sigma Aldrich) at a final concentration of 100 μg/ml before collection by centrifugation (6000 × g, 5 min) at 4°C. Collected cells were flash frozen in liquid nitrogen.
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| Growth protocol |
Overnight stationary cultures of wild type S. Typhimurium (Salmonella enterica serovar Typhimurium - strain SL1344) grown in LB media at 37°C with agitation (200 rpm) were diluted at 1:200 in LB and grown until they reached and OD600 of 0.5 (i.e., logarithmic (Log) phase grown cells).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The frozen pellet of a 50 ml culture was re-suspended and thawed in 1 ml ice-cold lysis buffer for polysome isolation (10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris.HCl pH 8.0, 20 U/ml of RNase-free DNase I (NEB 2U/µl), 1mM chloramphenicol (or 300µg/ml), 20 µl/ml lysozyme (50mg/ml in water) and 100u/ml SUPERase.In™ RNase Inhibitor (Thermo Fisher Scientific, Bremen, Germany)), vortexed and left on ice for 2 min with periodical agitation. Subsequently, the samples were subjected to mechanical disruption by two repetitive cycles of freeze-thawing in liquid nitrogen, added 5mM CaCl2, 30µl 10% DOC and 1 × complete and EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and left on ice for 5 min. Lysates were clarified by centrifugation at 16,000 x g for 10 min at 4°C. For the monosome sample, the supernatant was subjected to MNase (Roche diagnostics Belgium) digestion using 600 U MNase (about~ 1000 U per mg of protein). Digestion of polysomes proceeded for 1h at 25°C with gentle agitation at 400 rpm and the reaction was stopped by the addition of 10 mM EGTA. Next, monosomes were recovered by ultracentrifugation over a 1 M sucrose cushion in polysome isolation buffer without RNase-free DNase I and lysozyme, and with 2 mM DTT added using a TLA‐120.2 rotor for 4 hr at 75,000 rpm and 4°C.
For the selective purification of monosomes from polysomes (polysome sample), the supernatant was resolved on 10-55% (w/v) sucrose gradients by centrifugation using an SW41 rotor at 35,000 rpm for 2.5 hr at 4°C. The sedimentation profiles were recorded at 260 nm and the gradient fractionated using a BioComp Gradient Master (BioComp) according to the manufacturer's instructions. Polysome-enriched fractions were pooled and subjected to MNase digestion and monosome recovery as described above.Ribosome-protected mRNA footprints with sizes ranging from 26-34 nucleotides were selected and processed as described previously [23]with some minor adjustments as described
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
polysome bound mRNA footprints
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| Data processing |
The Ribosome profiling and RNA-seq sequenced reads were trimmed for adaptor sequences using fastx_clipper 0.0.14.
For the Ribo-seq reads the trimmed reads were sequentially aligned using Bowtie v0.12.7 to the Salmonella enterica rRNA and tRNA allowing for one mismatch. Reads aligning to any of these indices were discarded.
The trimmed RNA-seq and remaining Ribo-seq reads were then aligned using Bowtie V0.12.7 to the Salmonella enterica subsp. Enterica Typ str. SL1344 genome using parameters -v1 -m2 -k1.
The ribosome occupancy position was determined using the 5' end of the reads. For each footprint read the genomic position corresponding to the 5' end of the read was counted. Scores therefore represents the number of reads alignments attributed to each genomic position.
Genome_build: GCA_000210855.2
Supplementary_files_format_and_content: Bedgraph files were generated for the sense and antisense seperately: The positional scores represent the number of reads aligned to each genomic position.
Supplementary_files_format_and_content: Bed files were generated using bedtools. The scores represents the number of reads aligned to the region.
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| Submission date |
Dec 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Petra Van Damme |
| E-mail(s) |
Petra.VanDamme@UGent.be
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| Phone |
+32 9 264 9279
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| Organization name |
University of Gent
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| Department |
Department of Biochemistry
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| Lab |
Kris Gevaert Lab
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| Street address |
Albert Baertsoenkaai 3
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| City |
Gent |
| State/province |
East Flanders |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL20056 |
| Series (1) |
| GSE91066 |
REPARATION: Ribosome Profiling Assisted (Re-) Annotation of Bacterial genomes |
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| Relations |
| BioSample |
SAMN06127035 |
| SRA |
SRX2407648 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2420419_2382_PVD0008-Salmonella-poly_S2_AntiSense.bedgraph.gz |
4.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM2420419_2382_PVD0008-Salmonella-poly_S2_Sense.bedgraph.gz |
4.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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