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| Status |
Public on Aug 18, 2015 |
| Title |
clpP NO 1 |
| Sample type |
SRA |
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| Source name |
ΔclpP_DPTA treated
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: [Delta]lpP
treated with: 250 μM DPTA
sample type: bacterial liquid cell culture
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| Treatment protocol |
Immediately after inoculation of cells into the bioreactor, the culture was either treated with 250 μM DPTA, or an equal volume of only the DPTA solvent (20 μM NaOH) as an untreated control.
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| Growth protocol |
E. coli from a -80°C frozen stock were grown in LB for 4 h (37°C and 250 r.p.m.), and MOPS minimal media (Teknova) + 10 mM glucose for 16 h (37°C and 250 r.p.m.). The overnight culture was inoculated into 20 ml of fresh MOPS + glucose media in a 250 ml baffled shake flask, and grown at 37°C and 250 r.p.m. until OD600 = 0.2. The culture was centrifuged for 3 min at 15,000 r.p.m., resuspended in fresh pre-warmed (37°C) MOPS + glucose, centrifuged for an additional 3 min at 15,000 r.p.m., and again resuspended in pre-warmed MOPS + glucose. The resuspension was delivered to 10 ml fresh pre-warmed (37°C) MOPS + glucose in a stirred, aerobic batch bioreactor to a final OD600 of 0.05.
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| Extracted molecule |
total RNA |
| Extraction protocol |
10 min after inoculation & treatment, 3 ml of culture was removed and immediately mixed with 6 ml of RNAprotect Bacteria Reagent (Qiagen). Purification was performed using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. Briefly, after 5 min incubation with the RNAprotect reagent, the sample was centrifuged for 10 min at 5000×g, and the supernatant carefully discarded. Cell pellets were stored at −80 °C until all replicates and conditions for that particular experiment were completed, allowing simultaneous purification of all samples. To eliminate potential DNA contamination, an on-column DNase digestion was performed using RNase-free DNase I (Qiagen).
Half a microgram of total RNA from each sample was subject to ribosomal RNA depletion using RiboZero Bacteria kit (Illumina, CA) and then converted to strand-specific RNA-seq library using the automated Apollo 324TM NGS Library Prep System and associated kits (Wafergen, CA) according to the manufacturer’s protocol, utilizing different DNA barcodes for each library. The libraries were examined on Bioanalyzer (Agilent, CA) DNA HS chips for size distribution, and quantified by Qubit fluorometer (Invitrogen, CA). The 12 RNA-seq libraries were pooled together at equal amounts and sequenced on Illumina HiSeq 2500 in Rapid mode as one lane of single-end 67 nt reads following the standard protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
base-calling was performed with RTA version 1.17.21.3
Split multiplexed single-end sequencing data using a separate index read (standard Illumina TruSeq protocol), reads were mapped to the E. coli MG1655 genome (NCBI NC_000913.3) using TopHat (v2.0.9), and counted for each gene using htseq-count (version 0.6.0) from the HTSeq Python package .
Raw gene counts were analyzed and evaluated for differential expression in R (version 3.1.0) using DESeq2 (version 1.4.5).
Genome_build: NCBI NC_000913.3
Supplementary_files_format_and_content: text files containing the raw counts for each gene; xlsx containing summary DESeq2 output
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| Submission date |
Aug 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark Brynildsen |
| Organization name |
Princeton University
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| Department |
Chemical and Biological Engineering
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| Lab |
Mark P. Brynildsen
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| Street address |
25 William Street
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| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
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| Platform ID |
GPL18956 |
| Series (1) |
| GSE60522 |
An ensemble-guided approach identifies ClpP as a major regulator of transcript levels in nitric oxide-stressed Escherichia coli |
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| Relations |
| BioSample |
SAMN02998037 |
| SRA |
SRX683466 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1481606_clpPNO1.txt.gz |
20.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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