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| Status |
Public on Jul 20, 2014 |
| Title |
LB-pH9 |
| Sample type |
SRA |
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| Source name |
whole organisms
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| Organism |
Escherichia coli O157:H7 |
| Characteristics |
strain: EDL933
growth phase: late logarthmic, early stationary phase
growth condition and treatment: Tenfold diluted LB medium at alkaline pH was buffered with 10 mM CHES and the pH was adjusted to 9.0 at 37°C and was filter sterilized (LB-pH9). After 7 h, the cells reached 1.5 x 108 cfu/ml and were harvested.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol, rRNA was removed with the Ribominus kit (life technologies).
The read fragments were phosphorylated and the library was constructed using the TruSeq smallRNA sample prep kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
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| Description |
rRNA depleted
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| Data processing |
SOLiD output as QUAL and CSFASTA files was converted to FASTQ with Galaxy. We mapped SOLiD and Illumina FASTQ files to the reference genome using Bowtie (settings for SOLiD data: 28 nt seed length, maximal two mismatches in the seed, a maximal threshold of 70 for the sum of the quality values at mismatched positions; Illumina data: 20 nt seed length, 0 mismatches in the seed) implemented in Galaxy. Using Samtools output SAM files were filtered for mappable reads only. We further converted SAM files to BAM files and indexed them to create BAM.BAI files.
The number of reads were normalized to reads per kilobase per million mapped reads (RPKM). Using this method, the number of reads is normalized with respect to the sequencing depth and the length of a given gene. For determination of counts and RPKM values, BAM files were imported into R (R Development Core Team) using Rsamtools. For further processing, the Bioconductor packages GenomicRanges and IRanges were used. Gene locations were determined by RefSeq and GenBank PTT files. The locations of the 16S rRNA and 23S rRNA are given by the RNT file from RefSeq. The method countOverlaps of IRanges was used to determine the remaining reads overlapping a 16S or 23S rRNA gene. We discarded these reads from further analysis due to the artificial removal of these rRNAs using the Ribominus kit as described above. countOverlaps is also used to determine the number of reads overlapping a gene on the same strand (counts). With these counts, we generated the RPKM values. For the value “million mapped reads”, the number of reads mapped to the genome minus the reads overlapping a 16S or 23S rRNA gene were used (see above). The differential gene expression was analyzed with the Bioconductor package edgeR (version 3.2.3) using the counts.
Genome_build: Escherichia coli (str. O157H7 substr. EDL933: 259
Supplementary_files_format_and_content: The xlsx file contains information for each annotated gene in each condition. For each gene, the first number indicates the logFC of a certain condition compared to LB; RPKM values are shown in parentheses. The magnitude of the absolute value of logFC is indicated by shades of grey. Significantly differentially expressed genes are in bold (i.e., p values ≤ 0.05 in edgeR).
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| Submission date |
Jun 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard B. Landstorfer |
| E-mail(s) |
Richard.Landstorfer@wzw.tum.de
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| Organization name |
Technische Universität München
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| Department |
Chair for Microbial Ecology
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| Street address |
Weihenstephaner Berg 3
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| City |
Freising |
| ZIP/Postal code |
85354 |
| Country |
Germany |
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| Platform ID |
GPL17333 |
| Series (1) |
| GSE48199 |
Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 (EHEC) under eleven different environmental conditions |
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| Relations |
| BioSample |
SAMN02211806 |
| SRA |
SRX312990 |
| 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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