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| Status |
Public on Jan 15, 2018 |
| Title |
Mixed |
| Sample type |
SRA |
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| Source name |
Rat mucosa inoculated with Mixed culture
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: SD
tissue: Middle ear Mucosa
inoculated with: Mixed culture (MRSA+PA)
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| Treatment protocol |
The bacterial cell suspensions were prepared in TSB medium. 50 µL cell suspension containing 1×107cfu of Staphylococcus aureus (MRSA) were injected into the middle ear cavity through the tympanic membrane of the right ear using a tuberculin syringe and a 27-gauge needle.
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| Growth protocol |
The animals were euthanized using a 1:1 combination of nitrous-oxide and oxygen. After one week of inoculation, the rats were sacrificed using carbon-dioxide inhalation, and the bulla was aseptically acquired. Bulla were immediately dissected and middle ear was exposed and preserved in RNA latter (Qiagen, Hilden, Germany). The mucosa membrane of the bulla preserved in RNA latter solution was carefully scraped and preserved in fresh RNA latter solution till RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. RNA was quantified by Nano-drop, and the RNA quality was assessed by analysis of rRNA band integrality on an Agilent RNA 6000 Nano kit (Agilent Technologies, Palo Alto, CA, USA).
Ahead of cDNA library construction, the 2ug of total RNA and magnetic beads with Oligo (dT) were used to enrich poly (A) mRNA. Then, the purified mRNAs were disrupted into short fragments, and the double-stranded cDNAs were immediately synthesized. The cDNAs was subjected to end-repair, poly (A) addition, and connected with sequencing adapters using the TruSeq RNA sample prep Kit (Illumina, CA). The suitable fragments automatically purified by BluePippin 2% agarose gel cassette (Sage Science, MA) were selected as templates for PCR amplification. The final library sizes and qualities were evaluated electrophoretically with an Agilent High Sensitivity DNA kit (Agilent Technologies, CA) and the fragment was found to be between 350–450 bp. Subsequently, the library was sequenced using an Illumina HiSeq2500 sequencer (Illumina, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Low quality reads were filtered according to the following criteria; reads contain more than 10% of skipped bases (marked as ‘N’s), reads contain more than 40% of bases whose quality scores were less than 20 and reads whose average quality scores of each read is less than 20. The whole filtering process was performed using the in-house scripts. Filtered reads were mapped to the human reference genome (Ensembl release 72) using the aligner STAR v.2.3.0e.
Gene expression level was measured with Cufflinks v2.1.1 using the gene annotation database of Ensembl release 72. Non-coding gene region was removed with-mask option. To improve the accuracy of measurement, multi-read-correction and fragbias- correct options were applied and all other options were set to default values. For differential expression analysis, gene level count data were generated using HT Seq-count v0.5.4p3 tool with the option "-m intersection-nonempty” and –r option considering paired-end sequence.
Based on the calculated read count data, differentially expressed gene (DEG) were identified using the R package called TCC. TCC package applies robust normalization strategies to compare tag count data. Normalization factors were calculated using the iterative DEGES/edgeR method. Q-value was calculated based on the p-value using the p.adjust function of R package with default parameter settings. Differentially expressed genes were identified based on the q-value threshold less than 0.05.
Genome_build: human reference genome (Ensembl release 72)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample, fold change with respect to control.
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| Submission date |
Feb 09, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mukesh Kumar Yadav |
| E-mail(s) |
mukiyadav@gmail.com
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| Organization name |
Korea University
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| Department |
Institute for Medical Device Clinical Trials
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| Street address |
148
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| City |
Seoul |
| State/province |
Seoul |
| ZIP/Postal code |
152703 |
| Country |
South Korea |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE94722 |
The differential gene expressions of rat mucosa colonized with single or multi-species of MRSA or PA were studied using RNA-sequencing of total transcriptome. |
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| Relations |
| BioSample |
SAMN06317446 |
| SRA |
SRX2546938 |
| 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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