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| Status |
Public on Jan 30, 2017 |
| Title |
DMS-seq 95°C in vitro WT |
| Sample type |
SRA |
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| Source name |
bacteria
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655
treatment: NA
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| Growth protocol |
All cultures were based on MOPS media with 0.2% glucose (Teknova), with full supplement (Neidhardt et al., 1974) minus methionine. An overnight liquid culture was diluted 400-fold into 200 ml fresh media. The culture was kept in a 1 liter flask at 37°C with aeration until OD420 reached 0.4.
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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 37°C 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 4°C.
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 157, 624-635 (2014).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
DMS-modified mRNA
DMS_95_WT_vitro
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| Data processing |
Library strategy: DMS-seq
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. For ribosome footprints and mRNA-seq, the position of each alignment is distributed into several nucleotides in the center of each read. For each 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. For DMS-seq, the position of each alignment was the position immediately 5' of the 5' end of the read.
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 |
Feb 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Yan Zhang |
| E-mail(s) |
yan.jenny.zhang@gmail.com
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| Organization name |
UC San Francisco
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| Department |
Microbiology and Immunology
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| Lab |
Carol Gross
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| Street address |
600 16th Street
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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) |
| GSE77617 |
Operon mRNAs are organized into ORF-centric structures that predict translation efficiency |
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| Relations |
| BioSample |
SAMN04457990 |
| SRA |
SRX1562742 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2055262_DMS_95_WT_vitro_for.wig.gz |
1.6 Mb |
(ftp)(http) |
WIG |
| GSM2055262_DMS_95_WT_vitro_rev.wig.gz |
1.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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