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| Status |
Public on Mar 20, 2020 |
| Title |
WT rep2_37°C |
| Sample type |
SRA |
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| Source name |
WT_37°C
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| Organism |
Escherichia coli |
| Characteristics |
genotype/variation: MG1655 (rph1 ilvG rfb-50)
treatment: Grown at 37°C, harvested at OD600nm 0.3-0.35
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| Treatment protocol |
The pellets were washed once with cold TES buffer (100 mM NaCl, 1 mM EDTA, 10 mM Tris hydrochloride [pH 7.4]), recentrifuged, and cells snap-frozen in liquid nitrogen and stored at −85 °C.
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| Growth protocol |
The pre-cultures were grown in LB medium at 37°C. Triplicate cultures of the wild-type and ∆elyC mutant strains were grown in 250 ml LB 1% NaCl liquid media, 250 rpm, at 21°C and 37°C to early-log phase (OD600 of 0.30 to 0.35). The samples were immediately centrifuged (6000 g) at 4 °C for 5 min.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using the RNeasy Mini Kit (QIAGEN) with the RNAprotect Bacteria reagent (QIAGEN) according to the manufacturer’s protocol. DNA was removed using RNase-Free DNase set from QIAGEN. The RNA was finally resuspended in RNase-Free Water (QIAGEN) and RNA concentration was quantified using the NanoDrop ND-1000 at 260 nm. RNA Ribosome Integrity Numbers (RIN) and purity of the RNA samples were assessed using the Agilent Bioanalyzer 2100 and RNA 6000 Pico Kit. The RIN numbers for all the twelve samples were 9.3 to 10. The rRNA depletion was performed using the Ribo-Zero Magnetic Gold Kit for Gram-negative Bacteria (Illumina).
RNAseq libraries were prepared using KAPA RNA stranded Kit (Roche-Nimblegen). Libraries were assessed and quantified by BioAnalyzer using the High Sensitivity DNA Kit (Agilent). Sequencing was performed on an Illumina HiSeq2000 machine, in 75-nucleotide paired-end mode.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
37c_wt2
HI.2835.005.Index_4.IK_37C-wt2
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| Data processing |
Poor quality reads were trimmed out with Trimmomatic software. The quality of the reads, before and after trimming, was assessed with FASTQC version 0. 11. 5.
The reads were aligned to the E. coli sub-strain MG1655 reference genome (GCA000801205.1) with Bowtie2 version 2. 2. 9. The reference genome used for the alignment, and the GTF annotation file used for counting the number of reads aligned to each feature, were downloaded from Ensembl (release 27). The aligned data saved as BAM files. The raw alignment read counts for each gene were calculated using featureCounts 1.5.1.
Normalization and differential expression (fold change /Log2 fold change) of genes from the raw alignment counts were determined with a significant p-values (< 0.05) using the DESeq 2. 1. 14.0 in the R program.
Genome_build: GCA000801205.1
Supplementary_files_format_and_content: The output from DESeq2, including the raw counts, normalized relative to the total number of reads. Differentially expressed genes were determined with a p-value (< 0.05), and as part of the multiple comparison testing, significant Benjamini-Hochberg adjusted p-values (p-adj) < 0.05 (5% false discovery rate) were considered. In addition to the counts normalized relative to the library size, the genome-wide occupancy (quantitation) was also computed into density units of fragments per kilobase of exon model per million reads mapped (FPKM).
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| Submission date |
Dec 09, 2019 |
| Last update date |
Mar 20, 2020 |
| Contact name |
Fardin Ghobakhlou |
| Organization name |
University of Montreal
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| Department |
Microbiology , Infectiology and Immunology
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| Lab |
Dr. Paradis-Bleau
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| Street address |
Pavillon Roger-Gaudry, S-640 2900 Boulevard Edouard-Montpetit, Montréal, QC
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3T 1J4 |
| Country |
Canada |
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| Platform ID |
GPL14548 |
| Series (1) |
| GSE141694 |
Phenotypic and transcriptomic characterization of ElyC-defective Escherichia coli cells reveal the importance of ElyC in cell envelope biosynthesis at optimal and sub-optimal temperatures |
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| Relations |
| BioSample |
SAMN13514097 |
| SRA |
SRX7291199 |
| 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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