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| Status |
Public on Apr 30, 2021 |
| Title |
Co6hBR2 |
| Sample type |
SRA |
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| Source name |
WT (bioreplicate 2)
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: str 168
genotype/variation: WT
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| Treatment protocol |
Bacillus subtilis 168 PcomG‐gfp chloramphenicol resistant variant PcomG‐gfp was grown as described in growth conditions Samples for protein analysis and RNA‐seq analysis were taken at 5.5 and 6.5 h respectively. One hour of sorting through FACS yields approximately 3 × 10^7 GFP‐negative (non‐competent cells) and 1.5 × 10^7 GFP‐positive (competent) cell
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| Growth protocol |
The following competence medium was used: 18 ml demineralized water, 2 ml 10× competence medium stock [0.615 M K2HPO4•3H2O, 0.385 M KH2PO4, 20% glucose, 10 ml 300 mM Tri‐Na‐citrate, 1 ml 2% ammonium ferric citrate, 1 g casein hydrolysate (oxoid), 2 g potassium glutamate], 100 μl 2 mg ml-1 tryptophan, 67 μl 1 M MgSO4 (Spizizen, 1958; Konkol et al., 2013). Strains were streaked out from -80 stocks on Luria Bertani (LB) agar plates with antibiotics and grown overnight at 37°C. A single colony (sc) was diluted 1000× in PBS or 1× Spizizen solution 100 μl of the sc colony solution was added to 20 ml medium in 100 ml Erlenmeyer flasks and grown at 37°C 220 rpm. Exponential/early stationary overnight cultures were diluted to an OD600 of 0.05 in 20 ml medium without antibiotics. Antibiotic concentrations used were chloramphenicol (cm) 5 μg ml-1, spectinomycin (sp) 50 μg ml-1, erythromicin (ery) 0.5 μg ml-1, and lincomycin 12.5 μg ml-1. Growth conditions in CDSM (Vasantha and Freese, 1980; Hageman et al., 1984) + alanine (10 mM) + tryptophan 1 mM. Strains were grown overnight at 37°C on LB agar + chloramphenicol (control) or chloramphenicol + erythromycin (BFA1698), single colonies were diluted and incubated in 2 ml LB 37°C 220 rpm in test tubes. The diluted cultures were mid‐exponential after overnight growth. The overnight cultures were diluted to OD600 0.05 in 2 ml CDSM + alanine + tryptophan and chloramphenicol (control) or chloramphenicol + erythromycin (BFA1698) in test tubes and grown to mid‐exponential growth at 37°C 220 rpm. Cultures were diluted to OD600 0.1 in 100 μl CDSM + alanine + tryptophan without antibiotics in a 96 wells plate and grown at 37°C, 240 rpm, 10 min measuring interval for 20 h in a Thermo Fisher Varioskan Lux. The remainder of the cultures was grown for 24 h after which the cultures were kept in the dark at 4°C without shaking for 4 days. Spores were harvested by centrifugation at 10 000 g and washed 3× with double distilled water. The spore crops were diluted to the same OD and heated for 10 min at 80°C and dilutions were plated on LB agar with chloramphenicol and grown overnight at 37°C. Colonies were counted and measured with ImageJ. Statistics were done in Sigmaplot using a Rank Sum Test.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell were lysed using glassbead and totRNA was isolated using phenol/chloroform and ethanol precipitation
Standard library preparation protocol of BGI for Illumina RNA-Seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
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| Description |
2526_2-Co6hBR2
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| Data processing |
mapping of reads was done using: bowtie2 --local using Escherichia coli K12 as template genome
SAM files were converted to BAM files using SAM tools
BAM files were converted to RPKM values using BED-tools
Count values were normalized using the TMM (Trimmed Median Mean) method in T-REx (doi: 10.1186/s12864-015-1834-4)
Genome_build: Bacillus subtilis subsp. subtilis str. 168, circular BCT 15-JAN-2018, AL009126.3
Supplementary_files_format_and_content: Tab delimited text file
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| Submission date |
Apr 29, 2021 |
| Last update date |
Apr 30, 2021 |
| Contact name |
Anne de Jong |
| E-mail(s) |
anne.de.jong@rug.nl
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| Phone |
+31 50 363 2047
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| Organization name |
university of Groningen
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| Department |
Molecular Genetics
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| Street address |
Nijenborgh 7
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| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
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| Platform ID |
GPL23851 |
| Series (1) |
| GSE173540 |
Analyses of competent and non‐competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence |
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| Relations |
| BioSample |
SAMN18920643 |
| SRA |
SRX10709000 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5269464_Co6hBR2.rpkm.gz |
116.8 Kb |
(ftp)(http) |
RPKM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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