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Status |
Public on May 20, 2024 |
Title |
Sample Stop50_2 |
Sample type |
SRA |
|
|
Source name |
cell line
|
Organisms |
Mus musculus; Murid gammaherpesvirus 4 |
Characteristics |
tissue: cell line cell line: NIH 3T12 cell type: fibroblast genotype: WT treatment: MHV68 ORF50.stop
|
Treatment protocol |
NIH 3T12 fibroblasts were infected with ORF50.stop or WT MHV68 at MOI 3 for 18 hours.
|
Growth protocol |
NIH 3T12 (ATCC CCL-164) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 ug/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamine to form complete DMEM (cDMEM).
|
Extracted molecule |
polyA RNA |
Extraction protocol |
Total RNA was extracted using Trizol following manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Stranded, Poly A selection.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
Reads were trimmed for adapters and low-quality bases using the Cutadapt v1.18. Reads were aligned to our hybrid genome (mm10_MHV68YFP_Krug.fa) using STAR v2.4.2a in 2-pass mode. Gene and isoform expression was quantified using RSEM v1.3.3 with our custom GTFs (see below).. Low count genes were filtered prior to CPM and quantile normalization using Limma voom followed by differential expression of genes analysis. To map both host and viral reads, the viral genome was added as an extra chromosome to the mm10 genome, and the viral GTFs appended to the M21 mouse gene annotation (see provided FA and GTF files). Assembly: The host genome was mm10 using the M21 annotation, while our custom viral genome consisted of MHV68 with our YFP inserted into it. Two GTFs for the viral genome were created, one with full ORF annotaions (Krug_Ov) and one where overlapping sections were removed (Krug_NoOv). The data were analyzed with both GTFs for comparison. Supplementary files format and content: The FASTA (.fa) file provided contains our hybrid genome consisting of the mm10 mouse genome concatenated to our viral genome represaented as its own chromosome, which is MHV68 with our YFP marker inserted into it. Supplementary files format and content: The GTF (.gtf) files provided are for our Non-Overlapping (Krug_NoOv) gene annotation (where ORFs are trimmed to remove overlapping regions to make conservative expression estimates) and Overlapping (Krug_Ov) gene annotation, with full-length ORFs. Supplementary files format and content: The TXT (.txt) files provided are RSEM CPM TMM counts for the expression of our samples aligned against our hybrid genomes (Krug_NoOv and Krug_Ov).
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Submission date |
Mar 17, 2023 |
Last update date |
May 20, 2024 |
Contact name |
Thomas Joshua Meyer |
E-mail(s) |
thomas.meyer@nih.gov
|
Organization name |
National Institutes of Health
|
Department |
National Cancer Institute
|
Lab |
CCBR
|
Street address |
41 Center Drive, Building 41, Room B620
|
City |
Bethesda |
State/province |
Maryland |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL33263 |
Series (1) |
GSE227602 |
MHV68 ORF50stop Vaccine Gene Expression |
|
Relations |
BioSample |
SAMN33799248 |
SRA |
SRX19705399 |