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Sample GSM5528490

Query DataSets for GSM5528490

Status

Public on Jan 02, 2022

Title

E_096h

Sample type

SRA

Source name

bacterial cells

Organism

Clostridium tetani

Characteristics

condition: high pH (7.8)
time point: 96 h
replicate: 1
study number: 2

Treatment protocol

Growth conditions included: (A) normal (34°C, pH 7.3, 150rpm stirring); (B) no agitation; (C) 36 °C; (D) 32 °C; (E) pH 7.8; (F) increased N2 flux in headspace

Growth protocol

Clostridium tetani batch cultures were grown at Sanofi-Pasteur under different small-scale batch fermentation conditions. The cultures were performed during two studies; the second study served as a confirmation as well as an expansion of the first study. In the first study, cultures were performed in duplicate. Bacterial samples were taken at five time points if possible.

Extracted molecule

polyA RNA

Extraction protocol

10mL samples of cell suspension were collected and centrifuged (5 min at 4500 g). After supernatant removal, cell pellet was put in suspension with 500 μL of fresh medium. For RNA preservation, 1 mL of RNA-protect Bacteria reagent (Qiagen) was added. After homogenization, samples were stored at 5 °C for 5 min to 1 hour and then stored at -70 °C until RNA extraction. After thawing, a maximum of 10^9 cells were collected for RNA extraction. Cell content was not enough for certain samples, in this case the totality of sample was used. RNA extraction and purification were performed by using the RiboPure™-Bacteria kit from Life technologies (ref. AM1925) and the RiboPure Bacteria procedure (Part Number 1925M Rev. D). RNA QC was performed to determine the concentration and the integrity (RIN number).
For the first study, RNA-Seq was performed at RIVM. Library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (Illumina). Additional reagents used were the Ribo-Zero rRNA removal kit for Gram-Positive Bacteria (Illumina), RNA clean & concentrator-5 (Baseclear), and the Baseline-ZERO DNase (Immunosource). Libraries combined with 1% PhiX Control v3 (Illumina) were sequenced using Illumina NextSeq 500 equipment and the NextSeq 500/550 High Output Kit v2.5. For the second study, RNA-Seq was performed at BaseClear, using their Illumina-based protocols for bacterial RNA-Seq.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

FASTQ sequence files were generated using the Illumina Casava pipeline version 1.8.3. Initial quality assessment was based on data passing the Illumina Chastity filtering.
Reads containing PhiX control signal were removed. Reads containing (partial) adapters were clipped up to minimum read length of 50bp.
The second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.10.0.
FASTQ sequence reads were trimmed to their first 50 nucleotides, in order to limit the chance of reads mapping to more than one gene.
Trimmed read sequences were mapped to the E88 chromosome and plasmid reference sequence using bowtie2 (version 2.2.4) with default settings, after which a table with the number of counts per gene was obtained using samtools (version 1.1).
Genome_build: NC_004557.1 (chromosome) and NC_004565.1 (plasmid)
Supplementary_files_format_and_content: Countdata.xlsx: count data per sample plus gene annotation

Submission date

Aug 19, 2021

Last update date

Jan 02, 2022

Contact name

Jeroen Pennings

E-mail(s)

Jeroen.Pennings@rivm.nl

Phone

+31 88 689 2214

Organization name

Natl. Inst. Public Health & Environment