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Status |
Public on Apr 24, 2014 |
Title |
ribosome profiling in rich defined media |
Sample type |
SRA |
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Source name |
bacteria
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Organism |
Escherichia coli |
Characteristics |
strain: MG1655 media: fully supplemented MOPS glucose media
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Growth protocol |
All cultures were based on MOPS media with 0.2% glucose (Teknova), with either full supplement (Neidhardt et al., 1974). An overnight liquid culture was diluted 400-fold into 200 ml fresh media. The culture was kept in a 2.8-liter flask at 37C with aeration (180 rpm) until OD600 reached 0.3.
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Extracted molecule |
total RNA |
Extraction protocol |
Extraction was performed as described in detail previously (Li et al., 2012; Oh et al., 2011). 200 ml of cell culture was rapidly filtered at 37C by passing through a nitrocellulose filter. Cell pellets was were rapidly collected using a pre-warmed metal table crumber, flash frozen in liquid nitrogen, and combined with frozen droplets of lysis buffer. Cells and lysis buffer were pulverized in 10 ml canisters (Retsch) pre-chilled in liquid nitrogen using Qiagen TissueLyser II. Pulverized lysate was thawed on ice and clarified by centrifugation at 4C. Ribosome protected mRNA fragments were size selected via gel purification, and ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified [Oh et al,. Cell 147, 1295 (2011)]. More at G.W. Li, D. Burkhardt, C.A. Gross, J. S. Weissman (Cell).
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
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Description |
ribosome protected mRNA fp_rdm_pooled_f.wig fp_rdm_pooled_r.wig
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Data processing |
Basecalls performed using Casava versions 1.6 or 1.7. Sequenced reads were trimmed for adaptor sequence. Trimmed reads were sequentially aligned using Bowtie v0.12.7 to E coli rRNA and noncoding RNA allowing one mismatch. Reads aligning to any of these indices were discarded. The remaining reads were aligned using Bowtie v0.12.7 against E. coli MG1655 genome using parameters -v1 -m2 -k1. Bowtie alignments against the E coli genome were converted to wiggle files. The position of each alignment is distributed into several nucleotides in the center of the footprint. For each footprint read, the center residues that are at least 10 nucleotides away from either ends were given the same score, which is weighted by the length of the fragment [Oh et al,. Cell 147, 1295 (2011)]. Scores therefore represent the number of read alignments attributed to each genomic position under each scoring scheme. Genome_build: NC000913.2 Supplementary_files_format_and_content: wiggle files with two columns: first column containing chromosome positions and second column containing the number of reads mapped to the position (see publication for details).
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Submission date |
Jan 02, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Gene-Wei Li |
E-mail(s) |
gene-wei.li@ucsf.edu
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Organization name |
UCSF
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Street address |
1700 4th Street, BH404
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City |
San Francisco |
State/province |
CA |
ZIP/Postal code |
94158 |
Country |
USA |
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Platform ID |
GPL14548 |
Series (1) |
GSE53767 |
Absolute quantification of protein production reveals principles underlying protein synthesis rates |
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Relations |
BioSample |
SAMN02566834 |
SRA |
SRX403935 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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