GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2411715

Query DataSets for GSM2411715

Status

Public on Mar 27, 2017

Title

chemostat STR-PFR culture PFR P1 25min, rep2

Sample type

SRA

Source name

chemostat STR-PFR culture

Organism

Escherichia coli

Characteristics

strain: K12 W3110
sample port: PFR P1
time: 25min
replicate: 2

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.

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

E14R025e05

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

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

Organization name

University of Stuttgart

Department

Institute of Biochemical Engineering