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| Status |
Public on Feb 03, 2023 |
| Title |
Porhyromonas gingivalis PG1236-37-deficient mutant NO stress [X1236-37_00_NO_A] |
| Sample type |
SRA |
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| Source name |
Porhyromonas gingivalis PG1236-37-deficient mutant NO stress
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| Organism |
Porphyromonas gingivalis |
| Characteristics |
growth stage: Fresh culture, OD600~0.3
strain: W83
cell type: bacteria
genotype: PG1236-37-deficient
treatment: DEA-treated, 15 mins
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| Treatment protocol |
At OD600~0.3 time point, the bacteria cultures were stressed with Diethylamine (DEA) NONOate (15µl, 24mM stock concentration) for the NO stress studies. Untreated wild type and mutant strains cell cultures grown under the same conditions were used as controls. Samples were taken after 15 mins of NO stress treatment and controls were taken from untreated cultures at the same time.
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| Growth protocol |
Fresh cultures of P. gingivalis strains (mutants and wild type) were grown from overnight cell cultures under anaerobic conditions at 37ºC in BHI broth. P. gingivalis strains were grown to early exponential phase (OD600:~0.3) in BHI broth under anaerobic conditions at 37ºC.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the NO-stressed samples and controls using a total RNA isolation kit (Promega, WI). Additional DNase treatment was carried out using DNase kit (Ambion, Austin, TX).
The library was generated using RiboMinus Transcriptome Isolation Kit (Life Technologies, CA) and NEXTflex RNA-Seq Kit (Bioo Scientific, TX) following the vendors’ protocols. Briefly, 2 μg total RNA (20 μl) was hybridized with 4 μl of RiboMinus probe in 100 μl hybridization buffer at 37 ºC for 5 min, which was then cooled on ice for at least 30 seconds. The sample was transferred into an Eppendorf tube containing RiboMinus Beads resuspended in 100 μl hybridization buffer, which was incubated at 37 ºC for 15 min to allow beads to bind with probes. The tube was placed on a magnetic separator for 1 min to pellet the rRNA-probe complex. The supernatant containing RiboMinus RNA was transferred into a new tube. The RiboMinus RNA was concentrated by ethanol precipitation and was resuspended in 14 μl nuclease free water. Five μl of NEXTflex RNA Fragmentation buffer (Bioo Scientific, TX) was mixed with the 14 μl RiboMinus RNA. The mixture was incubated at 95 ºC for 10 min and then chilled on ice immediately. The first and second strand cDNAs were synthesized following the Bioo Scientific protocol and the NEXTflex RNA-Seq barcode was ligated to double strand cDNA after the end repair and adenylation for multiplexing. The library was amplified by PCR.
RNA seq. The quality of each RNA-Seq library was checked using Agilent Bioanaylzer and DNA 1000 Nano chip and the library was quantified using Qubit (Life Technology, CA). The libraries (20 samples) were loaded into a single lane of Illumina V3 flow cell for cluster generation and the sequencing was done on Illumina HiScanSQ with 100 x 100 BP of paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiScanSQ |
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| Data processing |
The mapped reads were then processed using Cufflinks (http://cufflinks.cbcb.umd.edu/) for transcript assembly. Read counts for each annotated gene in the P. gingivalis W83 genome assembly version ASM758v1 were calculated, normalized as reads per million mapped reads (RPM), and then transformed into log2 scale. To avoid infinite values, 1 count was added to each gene before log2 transformation.
The quality of reads was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw reads were filtered by Trimmomatic-0.22 (http://www.usadellab.org/cms/index.php?page=trimmomatic), including removing: 1) reads with sequencing adapters; 2) the 5’-end 5 bases of each read; 3) reads with more than 20 bases of ‘N’ nucleotides; 4) reads with low quality; and 5) reads with length less than 36 bases after trimmed. Trimmed reads were mapped to P. gingivalis W83 genome (NCBI accession ID: NC_002950) using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). The mapped reads were then processed using Cufflinks (http://cufflinks.cbcb.umd.edu/) for transcript assembly. Read counts for each annotated gene in the P. gingivalis W83 genome assembly version ASM758v1 were calculated, normalized as reads per million mapped reads (RPM), and then transformed into log2 scale. To avoid infinite values, 1 count was added to each gene before log2 transformation. Differentially expressed genes (DEGs) were determined with a non-stringent p-value cutoff of ≤ 0.05 and a fold change (FC) ≥ 1.5 (both up regulated and down regulated) compared with wild type W83. The Gene Ontology (GO) functional clustering and pathway analyses were done using NCBI DAVID Bioinformatics Tool (http://david.abcc.ncifcrf.gov/home.jsp) and KEGG GENES Database (https://www.kegg.jp/kegg/genes.html).
Supplementary files format and content: The processed data file is an excel file "RNAseq data_CdhR involvement in Pg in NO stress.xlsx" that includes data on fold change in gene expression for controls and NO-stressed samples. Genes IDs, p values of test, log2 of fold change, fold change and description of the sample are listed in columns entitled "Gene symbol, p value, Log2 (Fold change), Fold change, and GroupA_B(A/B)".
Assembly: P. gingivalis W83 genome assembly version ASM758v1
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| Submission date |
Jan 30, 2023 |
| Last update date |
Feb 03, 2023 |
| Contact name |
Marie-Claire Boutrin |
| Organization name |
Oakwood University
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| Street address |
7000 Adventist Blvd NW
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| City |
Huntsville |
| ZIP/Postal code |
35896 |
| Country |
USA |
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| Platform ID |
GPL33075 |
| Series (2) |
| GSE224065 |
The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress [RNA-seq] |
| GSE224067 |
The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress |
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| Supplementary data files not provided |
| Raw data not provided for this record |
| Processed data are available on Series record |
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