|
| Status |
Public on Oct 04, 2021 |
| Title |
Mock-WT S6 |
| Sample type |
SRA |
| |
|
| Source name |
spleen
|
| Organism |
Rattus norvegicus |
| Characteristics |
condition: WT
|
| Treatment protocol |
Weanling WT and Ifnar1-/- rats of 21-25 days of age of either sex were infected with a single i.p. dose of KRV-UMass strain (1 X 107 PFU) on day 0. Spleens were harvested from mock-infected (i.e., injected with culture media) WT rats (n=2) and Ifnar1-/- rats (n=2) or KRV-infected WT rats (n=4) and Ifnar1-/- rats (n=4) at 5 dpi.
|
| Growth protocol |
LEW.1WR1 rats (RT1B/Du) were from Biomere (Worcester, MA). Animals were housed in viral antibody–free conditions, confirmed monthly to be serologically free of rat pathogens (Mordes, Leif et al. 2002), and maintained in accordance with institutional and national guidelines (Institute of Laboratory Animal Resources (U.S.) 1996). KRV-UMass strain was prepared and titered by plaque assay as previously described (Zipris, Hillebrands et al. 2003).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Spleens were homogenized in 1 mL TRIzol reagent using TissueRuptor (Qiagen) and total RNA was extracted following TRIzol method (Invitrogen). RNA concentrations were quantified using a NanoDrop spectrophotometer (NanoDrop).
Approximately 10 mg total RNA for each of the 12 rat samples was treated with TurboDNase (ThermoFisher Scientific) and ribosomal RNA (rRNA) depletion was performed using a Ribo-ZeroTM Gold rRNA removal kit (Illumina). For RNA-seq, we prepared strand-specific libraries by following the protocol from Zhang and colleagues (Zhang, Theurkauf et al. 2012). The quality of the prepared libraries was confirmed using the Advanced Analytical Technologies, Inc. Fragment Analyzer through the Molecular Biology Core Lab (UMass Medical School).
The 12 libraries were pooled and sequenced with paired end reads (75 bp each) using Illumina NextSeq 500 according to the manufacturer’s specifications.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
S6
Mock-infected
|
| Data processing |
All raw sequencing reads were processed using an in-house pipeline (Dolphin) at the University of Massachusetts Medical School.
The clean read pairs were aligned to the rat reference genome and rRNA sequences were filtered from the mapped reads.
The RNA-Seq by Expectation Maximization (RSEM) method was used to determine the expression levels of genes by providing mapped transcript counts in the RNA-seq reads (Li and Dewey 2011).
The differentially expressed genes (DEGs) were identified using DESeq2 and significant differences in genes were identified as fold change >2 between uninfected and virus-infected samples and if the adjusted P value (Padj) was <0.05 (Love, Huber et al. 2014).
|
| |
|
| Submission date |
May 10, 2018 |
| Last update date |
Oct 04, 2021 |
| Contact name |
Alper Kucukural |
| E-mail(s) |
alper.kucukural@umassmed.edu
|
| Phone |
7743124493
|
| Organization name |
UMass Medical School
|
| Department |
Program in Molecular Medicine
|
| Lab |
Biocore
|
| Street address |
364 Plantation Street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL20084 |
| Series (1) |
| GSE114322 |
Gene expression analysis of spleens from virus-infected rats |
|
| Relations |
| BioSample |
SAMN09112898 |
| SRA |
SRX4066023 |
