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Sample GSM4284336

Query DataSets for GSM4284336

Status

Public on Feb 15, 2020

Title

12BC_ATCC19406

Sample type

SRA

Source name

bacterial cells

Organism

Clostridium tetani

Characteristics

strain: ATCC19406
lab: BaseClear
replicate: NA

Growth protocol

C. tetani inoculums were propagated for 48 hrs in thioglycollate media containing resazurine (Biomerieux, Craponne, France) at 35 °C.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA purification was performed with GenElute bacterial genomic DNA Kit (Sigma, St Louis, Missouri, USA) using standard protocol. After harvesting, cell pellets were normalized at ~10^10 cells by centrifuging enough cell suspension at 12,000 g for 5 min. Cell pellet was re-suspended in 4 x 106 UI/mL lysozyme solution (Sigma) for 30 min at 37 °C. RNase solution (Sigma) was added and incubation was done at room temperature for 2 min. Then protein digestion was done by addition of a lysis solution containing proteinase K (Sigma) C for 10 min at 55 °C. Then samples were processed for DNA isolation. Quality control of each purified DNA was performed by determining the purity by a spectrophotometer (ratio A260/A280, ratio A260/A230) and double-stranded DNA quantification by Qubit fluorimeter (Fisher Scientific, Waltham, Massachusetts, USA).
For sequencing at BaseClear, samples were used for Illumina genomic Nextera XT library preparation (including quality control and quantification), followed by Illumina HiSeq 2500 125 bp paired end sequencing. At Sanofi Pasteur, samples were processed using Illumina MiSeq equipment and reagent kits, for paired-end 250 bp paired-end sequencing.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

Library strategy: DNA-seq
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).
Gene count data output was further analyzed in R software (version 3.5.1).
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

Jan 24, 2020

Last update date

Feb 15, 2020

Contact name

Jeroen Pennings

E-mail(s)

Jeroen.Pennings@rivm.nl

Phone

+31 88 689 2214

Organization name

Natl. Inst. Public Health & Environment