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| Status |
Public on Apr 21, 2018 |
| Title |
DMS-MaPSeq of purified cspA mRNA at 10°C with 0.1mM CspA protein, rep 4 |
| Sample type |
SRA |
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| Source name |
DMS-MaPSeq of purified cspA mRNA at 10°C with 0.1mM CspA protein
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
treatment: 0.1mM CspA protein
temperature: 10°C
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| Treatment protocol |
For in vivo DMS modification, 15 mL of E. coli culture was incubated with 750 µL DMS. Incubation was performed for 2 min at 37°C or for 45 min at 10°C. DMS was quenched by adding 30 mL 0°C stop solution (30% β-mercaptoethanol, 25% isoamyl alcohol), after which cells were quickly put on ice, collected by centrifugation at 8000 x g, 4 °C for 2 min, and washed with 8 mL 30% BME solution. Cells were then resuspended in 450 µL total RNA lysis buffer (10 mM EDTA, 50 mM sodium acetate pH 5.5), and total RNA was purified with hot acid phenol (Ambion).
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| Growth protocol |
All culture experiments were performed in MOPS medium supplemented with 0.2% glucose, 19 amino acids (without methionine), vitamins, bases and micronutrients (MOPS rich defined medium minus methionine, Teknova). Cells were grown in an overnight liquid culture at 37°C, diluted to an OD420 = .001 in fresh medium and grown until OD420 reached 0.4 where samples were collected. For 10°C samples, cultures were grown to OD420 = 1.1 at 37°C and cold shock was performed by mixing 70mL of 37°C culture with 130mL of 0°C media pre-chilled in ice-water slurry, with continued growth of the culture in a 10°C shaker.
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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; Rouskin et al., 2014). For ribosome profiling, 200 ml of cell culture was rapidly filtered 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. Lysate containing 0.5 mg of RNA was digested for 1 h with 750 U of micrococcal nuclease (Roche) at 25°C. The ribosome-protected RNA fragments were isolated using a sucrose gradient followed by hot acid phenol extraction.
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., 2011; Li et al., 2014; Rouskin et al., 2014).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
DMS-modified mRNA purified from in vitro transcription reaction
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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. 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.
For DMS-MaPSeq, reads were aligned to the 5’UTR of cspA using bowtie2 and an alignment seed of 12nt. Low sequence quality bases (Q score < 20%) and missing bases due to truncated reads were set to question marks. Reads that consisted of more than 20% question marks were filtered out. Only mutations that agreed between the forward and the reverse read were considered true mutations. Mutation rate per base was calculated as number of reads with a mutation at the base divided by total number of reads covering the base.
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).
Supplementary_files_format_and_content: Txt files are processed data for DMS-MaPseq with two columns: first column containing chromosome positions (relative to transcription start site (1) of cspA) and second column containing the mutation rate.
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| Submission date |
Jan 24, 2018 |
| Last update date |
Apr 21, 2018 |
| Contact name |
Yan Zhang |
| E-mail(s) |
[email protected]
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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 |
GPL17439 |
| Series (1) |
| GSE103421 |
A stress response that monitors and regulates mRNA structure is central to cold-shock adaptation |
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| Relations |
| BioSample |
SAMN08391110 |
| SRA |
SRX3596414 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2946871_DMS_MaP_cspA_5UTR_10C_100CspA_rep4.txt.gz |
967 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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