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| Status |
Public on Oct 28, 2023 |
| Title |
TM3_2_19_3_Lysozyme [10 µg ml-1] |
| Sample type |
SRA |
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| Source name |
whole cells
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: Bacillus subtilis W168 (BaSysBio)
genotype: trp+
growth phase: logarithmic phase
treatment: Lysozyme [10 µg ml-1]
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| Treatment protocol |
The B. subtilis W168 at exponential growth phase of OD600 = 0.4 were treated with bacitracin (50 µg ml-1), vancomycin (1 µg ml-1), tunicamycin (10 µg ml-1), phosphomycin (10 µg ml-1), penicillin G (10 µg ml-1), flavomycin (10 µg ml-1) and lysozyme (10 µg ml-1), respectively, for 10 min at 37°C, 220 rpm . Non-treated samples were used as control. Each of the treatments and control were prepared in triplicates.
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| Growth protocol |
Overnight culture of B. subtilis W168 was prepared in LB medium (Sigma L3522) at 37 °C, 220 rpm overnight. On the next day, 10 ml LB medium (Sigma L3522) was inoculated with overnight culture in a 100 ml flask 1:100 and grown at 37 °C, 220 rpm until OD600 = 0.4-0.5 as day culture 1. Subsequently, 200 ml LB (Sigma L3522) in a 2000 ml flask was inoculated with appropriate amount of day culture 1 to an OD600 of 0.01, and incubated at 37 °C, 220 rpm until OD600 reached to around 0.4 as day culture 2. Then, day culture 2 was split into 25 ml aliquots and either exposed to corresponding antibiotics or remained untreated for 10 min under shaking. After that, cells were immediately transferred into pre-cooled 50 ml falcon tubes in ice/NaCl bath (ice:NaCl=3:1) to stop induction and then centrifuged at 4 °C, 8000 rcf for 3 min. The falcons containing the cell pellets were then frozen in liquid nitrogen and kept at -80°C until further use.
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| Extracted molecule |
total RNA |
| Extraction protocol |
B. subtilis cells were disrupted using a micro-dismembrator. Total RNA isolation was performed with a phenol-chloroform isoamyl-alcohol (25:24:1, pH 5.5) extraction method.
rRNA was subtracted from the samples with the Illumina Ribo-Zero rRNA removal Kit (Bacteria) according to manufacturer instructions. The cDNA library was prepared using the NEB Ultra directional RNA library prep kit for Illumina according to instructions and sequencing was performed on an Illumina HiSeq3000 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
3549_BH
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| Data processing |
mapping was done with bowtie 2 (bowtie 2: 2.4.1)
unmapped reads were filtered with samtools (samtools: 1.10).
Mapped reads were sorted, and converted to bam with samtools (samtools: 1.10)
counting features with FeatureCounts on the opposite strand (subread: 2.0.1)
counts were normalized using DESeq2 and a differential gene expression was calculated (DESeq2: 1.28.0, r-base: 4.0.2)
The DESeq 2 comparisons were combined and enriched to an Excel sheet using in-house scripts(, which can be accessed on request). Condition TM3_2_05 was used as the reference point.
With bamCoverage from Deeptools, bam files were normalized and converted to BigWig files for forward and reverse strand (deeptools: 3.4.3)
Genome_build: Assembly ASM904v1 (RefSeq: GCF_000009045.1); Genome annotation (in GFF format) was gathered from BSGatlas (Version 1.0)
Supplementary_files_format_and_content: Counts and normalized counts in tab separated files. A differential gene expression xlsx sheet containing all genes with p-values, adjusted p-values, log2 fold-changes, and information extracted from the GFF file. BigWig files contain coverages for either forward or reverse strand
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| Submission date |
Oct 28, 2020 |
| Last update date |
Oct 28, 2023 |
| Contact name |
Thorsten Mascher |
| E-mail(s) |
thorsten.mascher@tu-dresden.de
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| Phone |
+49 351 463-40420
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| Organization name |
Technische Universität Dresden
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| Department |
Institute of Microbiology
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| Lab |
General Microbiology
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| Street address |
Zellescher Weg 20b
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| City |
Dresden |
| ZIP/Postal code |
01217 |
| Country |
Germany |
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| Platform ID |
GPL29318 |
| Series (1) |
| GSE160345 |
Comprehensive Transcriptional Profiling of the Cell Envelope Stress Response of Bacillus subtilis |
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| Relations |
| BioSample |
SAMN16583459 |
| SRA |
SRX9390686 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4871870_TM3_2_19_3.forward.bw |
2.3 Mb |
(ftp)(http) |
BW |
| GSM4871870_TM3_2_19_3.reverse.bw |
2.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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