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| Status |
Public on Jul 17, 2014 |
| Title |
mRNA-seq MicL-S t0 |
| 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
plasmid: pMicL-S
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| Treatment protocol |
At OD ~0.3, cultures were induced with 1mM IPTG for the appropriate length of time.
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| Growth protocol |
Bacteria were grown at 30C with shaking (~200 rpm) in fully supplemented MOPS glucose media (Teknova) to OD420 ~0.4. Cultures were split into 4 subcultures, and grown to OD ~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 and total mRNA fragments were size selected via gel purification. Fragments were 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 details at MS Guo, TB Updegrove, et al, Genes Dev. (2014).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Fragmented, then size selected 25-40bp fragments, for mRNA-seq
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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 genome using parameters -v 1 -m 1 --best. This allows one mismatch and reports only reads that align 1 or fewer times to the genome
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 |
Jul 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Carol A Gross |
| E-mail(s) |
cgross@cgl.ucsf.edu
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| Organization name |
UCSF
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| Street address |
600 16th St, S376
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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) |
| GSE58637 |
MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein |
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| Relations |
| BioSample |
SAMN02905015 |
| SRA |
SRX648120 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1430427_mrna-MicL-S0_f.wig.gz |
3.4 Mb |
(ftp)(http) |
WIG |
| GSM1430427_mrna-MicL-S0_r.wig.gz |
3.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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