GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4795111

Query DataSets for GSM4795111

Status

Public on May 05, 2021

Title

12-WT-STR-5min

Sample type

SRA

Source name

12-WT-STR-5min

Organism

Escherichia coli

Characteristics

time point: 5min
strain: MG1655
replicates: B
condition: nlim-nstarv
sample port: S

Growth protocol

A small amount of glycerol stock seed culture was spread onto 2xYT agar plates and incubated at 37 °C for 24 h. A single colony was picked to inoculate 500 ml baffled shaking flasks with 50 ml of preculture minimal media. Flasks were then incubated at 37 °C on an orbital shaker set to 150 rpm for 16 hours. In the next morning 500 µl of biosuspension were transferred to 1000 ml baffled shaking flasks containing 100 ml preculture minimal media and incubated at 37 °C on an orbital shaker set to 150 rpm for 8 hours. 50 ml of this culture were used to inoculate the bioreactor. Total volume in the bioreactor was 1.6 l after inoculation. Batch fermentation in the bioreactor ensued at 28-30 °C overnight. In the next morning feed and harvest trains were connected and a constant feed/harvest rate at 5.33 ml/min corresponding to a dilution rate of 0.2 h-1 established. After 25 h (five residence times) a reference sample was taken (t0). The plug-flow reactor was then connected to the primary reactor via a diaphragm metering pump effectively circulating about one-quarter of the total fermentation broth from the primary reactor through the plug-flow reactor and back into the stirred tank reactor. In the following 28 h samples were taken at predefined time points from the STR and the five PFR ports. After these 28 h the fermentation was aborted, and the final broth volume measured.

Extracted molecule

total RNA

Extraction protocol

0.5 ml broth was sampled from the bioreactor and directly flash-frozen in liquid nitrogen. Frozen broth was then stored at -70 °C until the day of RNA isolation. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Isolated RNA was DNAse treated and shipped to commercial sequencing partner GENEWIZ® on dry ice. Samples were treated for rRNA depletion, sequencing libraries prepared and Illumina HiSeq 2x150 bp sequencing performed. Raw FASTQ files were obtained for bioinformatic analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Escherichia coli WT MG1665

Data processing

Trimmomatic v. 0.32 (Bolger et al., 2014) was used to remove adapters and low quality reads (<Q20) checked by fastqc reports. Genes were aligned to the NCBI E. coli K12 MG1655 reference genome (RefSeq: NC_000913.3) using the RNA-sequencing aligner Bowtie2 v. 2.3.2.2 (Langmead and Salzberg, 2012). Aligned reads were counted for each gene based on the corresponding annotation available from NCBI for the chosen reference sequence applying HTseq-count v. 0.6.1 (Anders et al., 2014) in the union mode.
Differential gene expression analysis was performed with the R-package DeSeq2 v. 1.26.0 (Love et al., 2014) available from Bioconductor (Gentleman et al., 2004) (http://www.bioconductor.org). Prior to statistical analysis, all residual non-protein encoding RNA molecules (tRNA, rRNA and sRNA) were removed from the HTseq-derived raw count data and a non-speciﬁc ﬁlter was applied to remove low coverage genes with fewer than two counts per million (54 reads on average). All filtering steps caused deviations from the raw data of less than 6 %. Samples were grouped by replicates and an experimental design was chosen that used sample time and location (STR or PFR) as a combined environmental factor. To normalize the read counts for comparison purposes on sequencing depth and RNA composition, DESeq2 uses the median of ratios method to derive a scaling factor.. Resulting p-values were adjusted for multiple testing according to Benjamini and Hochberg (1995) to control the false discovery rate (FDR). Genes were identiﬁed as signiﬁcantly diﬀerentially expressed by applying FDR adjusted p-values < 0.01 and a log2 fold change ≥ |1|.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: xlsx file formate includes raw gene counts for each sample before and after filtering for low coverage genes. Additionally normalized gene counts are included.

Submission date

Sep 18, 2020

Last update date

May 05, 2021

Contact name

Julia Zieringer

Organization name

University of Stuttgart

Department

Biochemical Engineering

Street address

Allmandring.31