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Sample GSM6619226

Query DataSets for GSM6619226

Status

Public on Jan 05, 2023

Title

DC, rep1

Sample type

SRA

Source name

detached cells

Organism

Bacillus subtilis

Characteristics

cell type: detached cells
genotype: strain NDmed, wild-type
growth medium: B-medium

Growth protocol

Static liquid culture of 3mL of synthetic B-medium in 12-well microplates incubated at 30°C for 24 hours; 6 wells were used to collect one sample.

Extracted molecule

total RNA

Extraction protocol

Cells were collected in Eppendorf tubes containing 500µl TRIzol reagent.
After centrifugation (8,000 g at 4°C for 30 seconds), the supernatant was drowned off and the pellet was snap-frozen in liquid nitrogen and stored at -80°C for later use.
Pellets were washed with 1ml TE + 60µl EDTA (10mM Tris, 1mM EDTA, pH=8) followed by centrifugation (8,000 g at 4°C for 30 seconds).
Pellets were then suspended in 1ml TRIzol reagent before transfer to a Fastprep screw cap tube containing 0.4g of glass beads (0.1mm).
Cells were disrupted by bead beating for 40 seconds 6.5m/s with FastPrep-24 instrument (MP Biomedicals, United states).
The supernatant was transferred to an Eppendorf tube and chloroform (Sigma-Aldrich, France) was added in a ratio of 1:5, followed by centrifugation (8,000 g at 4°C for 15 minutes).
The chloroform step was repeated twice before transfer of the aqueous phase was transferred to new Eppendorf, where sodium acetate (pH=5.8) was added to a final volume of 10% and 500µl of isopropanol (Sigma-Aldrich, France). Samples were left overnight at -20°C.
After centrifugation (8,000 g at 4°C for 20 minutes), pellets were washed twice by ethanol 75% and centrifugation (8,000 g at 4°C for 15 minutes).
Then pellets were dried for 5 minutes under the hood. A RNA cleanup kit (Monarch RNA Cleanup Kit T2050, New England Biolabs, France) was used to further clean the RNA samples.
Nanodrop and Bioanalyzer instruments were used for quantity and quality controls.
Library construction and sequencing was performed by the I2BC platform (Gif-sur-Yvette, France).
Library construction was done using TruSeq Total RNA Stranded and Ribo-Zero Bacteria Illumina kits.
Sequencing was performed on an Illumina NextSeq 550 system using NextSeq 500/550 High Output Kit v2 to generate stranded single end reads (1 x 75bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

DC1

Data processing

Primary data processing was performed by I2BC platform and consisted of: demultiplexing (with bcl2fastq2-2.18.12), adapter trimming (Cutadapt 1.15), quality control (FastQC v0.11.5), mapping (BWA v0.6.2-r126) against NDmed genome sequence (NCBI WGS project accession JPVW01000000); and aggregation as read counts for 4028 (featureCounts).
Fragment counts normalised per kilobase of feature length per million mapped fragments (fpkm) were computed with DESeq2 v1.30.1 based on robust estimation of library size.
Assembly: NCBI JPVW00000000.1
Supplementary files format and content: tab-delimited text file
Supplementary files format and content: First 3 columns : locus tag in B. subtitilis NDmed genome (GenBank: JPVW00000000.1) ; corresponding locus tag in B. subtilis 168 reference genome (Genbank: AL009126.3) ; genes names in B. subtilis 168 as in Subtiwiki database (https://doi.org/10.1093/nar/gkab943).
Supplementary files format and content: Next 27 columns (9 cell types x 3 replicates) labelled from EX1.rawcounts to PL3.rawcounts : raw count of the number of reads mapped in each gene.
Supplementary files format and content: Last 27 columns (9 cell types x 3 replicates) labelled from EX1.log2fpkm to PL3.log2fpkm : log2(fpkm+5) for each gene.

Submission date

Oct 06, 2022

Last update date

Jan 05, 2023

Contact name

Pierre Nicolas

E-mail(s)

pierre.nicolas@inrae.fr

Phone

+33-1-3465-2894

Organization name

INRAE - Université Paris-Saclay