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Status |
Public on Mar 27, 2017 |
Title |
chemostat STR-PFR culture PFR P5 45min, rep2 |
Sample type |
SRA |
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Source name |
chemostat STR-PFR culture
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Organism |
Escherichia coli |
Characteristics |
strain: K12 W3110 sample port: PFR P5 time: 45min replicate: 2
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Treatment protocol |
Sampling by directly placing the culture sample into RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany), following short centrifugation the pellet was stored at −70°C.
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Growth protocol |
E. coli K12 substrain W3110 was used in this study. Bioreactor cultivations were performed using a two-compartment bioreactor system consisting of an STR (3L) with a recycle loop (PFR) in defined medium at pH7 and 37°C. Bioreactor cultivations were carried out with a minimal medium containing (per liter) 19.0 g glucose, 1.0 g NaH2PO4.· 2 H2O, 2.6 g K2HPO4, 3.8 g (NH4)2SO4, and a trace element solution (0.11 g Na3C6H5O7, 0.00835 g FeCl3· 6 H2O, 0.00009 g ZnSO4· 7 H2O, 0.00005 g MnSO4· H2O, 0.0008 g CuSO4· 5 H2O, 0.00009 g CoCl2· 6 H2O, 0.0044 g CaCl2· 2 H2O, 0.1 g MgSO4· 7 H2O). The STR-PFR was operated as a chemostat with ammonia as the growth-limiting substrate. First, a reference steady state (µ=0.2 h−1) was established in the STR and sampled three times during a 16 h period following establishment of the steady state. Then, the PFR was added and samples for RNA-sequencing were aquired.
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Extracted molecule |
total RNA |
Extraction protocol |
The RNeasy mini kit, with on column RNase-Free DNase I treatment, (both Qiagen, Hilden, Germany) was used for total RNA isolation (RNA ≥ 200 bases), according to the manufacturer’s protocol. RNA quality was determined by a Lab-on-a-Chip-System Bioanalyzer 2100 (Agilent, Boeblingen, Germany). First, ribosomal RNA was depleted from 1 μg of total RNA using the Ribo-ZeroTM Magnetic Kit (Bacteria) (Epicentre, Madison, WI, USA). Next, mRNA libraries were prepared using the TruSeq mRNA Library Prep Kit (Illumina, San Diego, CA, USA). mRNA libraries were prepared for sequencing using standard Illumina protocols.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
E14R025e06
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Data processing |
The bcl2fastq Conversion Software v. 1.8.4 from Illumina (http://support.illumina.com/downloads.html) was used to translate cluster intensity values into fastq files. Sequenced reads were trimmed for adaptor sequence using Cutadapt v. 1.8.3, then mapped to E. coli K12 substr. W3110 whole genome from NCBI (GenBank: AP009048.1) using the RNA-seq aligner STAR v.2.4.2a. Raw counts were derived for each gene based on the respective annotations available from UCSC genome browser (http://genome.ucsc.edu) for the chosen reference sequence applying HTseq-count v. 0.6.1 in the intersection-nonempty mode. HTseq-derived raw count data were used as input after a non-specific filtering step that removed residual rRNAs and tRNAs and low coverage genes with fewer than 2 counts per million (16-20 reads) in more than 25% of the dataset. Genome_build: ASM1024v1 (NCBI E. coli K12 subst. W3110 genome (GenBank: AP009048.1)) Supplementary_files_format_and_content: xlsx file format includes raw gene counts for each sample before and after filtering for low coverage genes
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Submission date |
Dec 01, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Joana Danica Simen |
E-mail(s) |
joana.simen@ibvt.uni-stuttgart.de
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Organization name |
University of Stuttgart
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Department |
Institute of Biochemical Engineering
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Street address |
Allmandring 31
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City |
Stuttgart |
ZIP/Postal code |
70569 |
Country |
Germany |
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Platform ID |
GPL18133 |
Series (1) |
GSE90743 |
RNA-seq of Escherichia coli under fluctuating ammonia availability (limitation-depletion) |
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Relations |
BioSample |
SAMN06102524 |
SRA |
SRX2391503 |
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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