SAMN06286527
External Id
Korea University
INSDC center name
2018-03-01T00:50:06.730Z
INSDC first public
2018-03-01T00:50:06.730Z
INSDC last update
live
INSDC status
Model organism or animal
NCBI submission model
Model.organism.animal.1.0
NCBI submission package
8 Weeks
http://purl.obolibrary.org/obo/UO_0000034
age
Pathogen-free Sprague-Dawley rats weighing 150-200 g were obtained from Koatech (Pyeongtaek, South Korea). Prior to experiment, animals were examined for abnormal middle ear and were housed in infection free zone for 2 weeks. Group 1, rats inoculated with SA, and Group 2 with media only (vehicle control). 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. The tympanic membrane was removed and ears were irrigated to remove the planktonic bacteria. The bulla from each group were dissected and cleaned. 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. 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). 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.
description
Rat middle ear
isolation source
Rattus norvegicus
http://purl.obolibrary.org/obo/NCBITaxon_10116
organism
Rat middle ear mucosa
sample type
male
http://purl.obolibrary.org/obo/PATO_0000384
sex
SD
strain
Rat middle ear mucosa colonized with SA (MRSA) and media only
tissue
Gene expression of SA (MRSA) colonized rat middle ear mucosa with respect to media only group.
title
Staphylococcus aureus (SA) injected in rat middle ear
treatment