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Sample GSM5219440

Query DataSets for GSM5219440

Status

Public on Jan 12, 2022

Title

C64 -1 [GLA-15_S18]

Sample type

SRA

Source name

Bacteria_C64

Organism

Bacillus cereus

Characteristics

strain: C64
replicate: 1
toxic: no

Growth protocol

Bacterial cultures were incubated in BHI medium at 30°C in microaerophilic condition (5% O2–15% CO2–80% N2) at pH 7 until entry into stationary growth phase. Samples were centrifuged at 12,000 g for 3 min at 4°C and placed immediately at -80°C until processing.

Extracted molecule

total RNA

Extraction protocol

The bacterial pellets were re-suspended with 200 μl of 10 mM Tris-HCl at pH 8 + 4 μl of lysozyme at 50 mg/ml and incubated at 37°C. Total RNA was extracted with the HPRNA kit (High Pure RNA Isolation Kit; Roche). Remaining traces of DNA were removed by two steps of DNase treatment (TURBO-DNAse Free kit, Ambion). The RNA integrity was measured by the RIN (RNA Integrity Number) obtained using the capillary electrophoresis Bioanalyser® (Agilent). RIN was calculated with the total RNA ratio of the area of ribosomal bands to the total area and the height of the 16S and 23S peaks. The RIN of samples were between 7 and 10. The mRNA were purified with the RiboZero Kit (Illumina).
Directional and paired libraries were prepared with the Illumina scriptseq kit and the sequencing was performed on an Illumina Nextseq machine.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

GLA-15_S18

Data processing

Low quality triming with sickle (options: -t sanger -x -n -q 20 -l 20)
Read mapping using bowtie2 version 2.2.6 (options: -N 1 -L,-0.2,-0.2)
Read counts on each allelic variant were obtained using HTSeq-count (version 0.6.1)
Differential expression analysis R library EdgeR" and associated "median ratio method" normalization procedure
EdgeR p-values were converted into q-values using R library "fdrtool"
Genome_build: In absence of whole genome sequences for the 15 strains, the cleaned reads were mapped against a repertoire of allelic variants for 23,815 genes aiming at accounting for the pangenome of B. cereus group. This repertoire was obtained by single-linkage clustering based on the results of an all-against-all blastn comparison (version 2.2.26, e-value cut-off 1e-5) [46] of 519,931 CDSs extracted from the 91 annotated complete genomes available at the time of analysis for B. cereus group in Genbank. Pairs of CDSs that aligned over at least 70% of the length of the shortest sequence and with at least 75% nucleotide sequence identity were grouped in the same cluster, which resulted in 23,815 clusters representing distinct genes.
Supplementary_files_format_and_content: csv file containing for each gene/sample raw data counts and rpkm

Submission date

Mar 30, 2021

Last update date

Jan 12, 2022

Contact name

Cyprien Guérin

E-mail(s)

[email protected]

Phone

+33-1-3465-2896

Organization name

INRAE