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| Status |
Public on May 17, 2021 |
| Title |
F. nucleatum delta-carR Rep 2 |
| Sample type |
SRA |
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| Source name |
bacterial culture
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| Organism |
Fusobacterium nucleatum subsp. nucleatum |
| Characteristics |
strain: ATCC 23726
treatment: Genes galK and carR deleted
growth phase: exponential growth
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| Growth protocol |
F. nucleatum were grown in tryptic soy broth (TSB) supplemented with 1% BactoTM peptone plus 0.25% freshly made cysteine (TSPC) or on TSPC agar plates in an anaerobic chamber (10% CO2, 10% H2 and 80% N2).
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| Extracted molecule |
total RNA |
| Extraction protocol |
F. nucleatum RNA was extracted using RNeasy Mini Kits (Qiagen) according to the manufacture’s protocol. Fusobacterial cell pellets from 3 ml log-phase culture (OD600 ~ 0.6) were harvested and resuspended into 200 μl of chilled 10 mM RNA-free TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Each suspension was transferred to a fast-protein tube (Q BIOgene), which contained 700 μl RLT buffer (RNeasy Mini Kit, Qiagen) and 7 μL β-mercaptoethanol (BME) (ThermoFischer Scientific. Cells were lysed by using Ribolyser (Hybaid), and supernatants were obtained by centrifugation at 13000 x g for 5 min at 4°C. RNA was then purified from the supernatant accordingly, and purified RNA was treated with DNAse (Qiagen) and cleaned by using an RNeasy clean up kit (Qiagen). Quality of RNA samples was determined RNA Integrity Number (RIN) values greater than 8 using an Agilent 2100 Bioanalyzer (Agilent Technologies).
2 µg total RNA from F.nucleatum parent and mutant strains were used. Stable RNAs were depleted with the RiboZero Kit according to manufacturer's instructions (Epicentre). Afterwards the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality with the Bioanalyzer (Agilent). Fragmentation of mRNA, reverse transcription to cDNA, adenylation of 3' ends, adapter ligation and PCR amplification were performed according to TrueSeq Stranded mRNA library instructions (Illumina). Prior to paired-end sequencing of the whole transcriptome cDNA libraries on an Illumina MiSeq, the quality and concentration of the libraries were checked using the Bioanalyzer (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Base calling with Illumina CASAVA
quality trimming with trimmomatic v0.36 from both ends with parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:39
reverse complementing of R1 reads
mapping with bowtie2 2.2.7, --ff read orientation, default parameters
converting SAM to BAM with samtools 1.2
visualization and read counting with readXplorer 2.2.3
Analysis of differentially transcribed genes with DESeq2 in default parameters
Genome_build: ASM17889v1
Supplementary_files_format_and_content: List_of_differentially_transcribed_genes.xlsx contains raw counts, normalized read counts and log2foldchanges and adjusted p-values for each gene.
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| Submission date |
Mar 02, 2021 |
| Last update date |
May 17, 2021 |
| Contact name |
Manuel Wittchen |
| Organization name |
Bielefeld University
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| Department |
Center for Biotechnology
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| Street address |
Universitaetsstr. 27
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| City |
Bielefeld |
| ZIP/Postal code |
33615 |
| Country |
Germany |
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| Platform ID |
GPL29799 |
| Series (1) |
| GSE168051 |
Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum |
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| Relations |
| BioSample |
SAMN18106697 |
| SRA |
SRX10206243 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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