|
| Status |
Public on Mar 07, 2016 |
| Title |
WT_080 |
| Sample type |
SRA |
| |
|
| Source name |
Wildtype (WT) Bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: CD8+ T cells
reporter: Blimp1/GFP
mrna extraction: Single-cell
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NP366+ CD8+ T cells were purified by flow cytometric sorting from day 9 HKx31-infected PR8-primed mixed bone marrow chimeras. Total RNA was prepared using a Qiagen RNeasy Plus Mini Kit (Qiagen) according to the manufacturers instructions. RNA-seq was carried out based on a previously published single-cell protocol (Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature protocols 5, 516-535, 2010). Samples were sequenced on a Solexa 1G Genome Analyzer instrument (Illumina) at the National Heart Lung and Blood Institute.
RNA libraries were prepared for sequencing using standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
RNA-seq reads were aligned to the GRCm37/mm9 build of the Mus musculus genome using the Subread aligner. Mapping quality score was used to break the tie when more than one best location was found for a read.
Genewise counts were obtained using featureCounts. Only reads overlapping with the last 3000 exonic bases (located at 3’ end) of each gene were counted because only these bases in the genes were sequenced in this experiment. All exonic bases will be used for read counting if a gene has a total exon length of less than 3000 bases. NCBI RefSeq mouse annotation build 37.2 was used in the analysis.
Genes were filtered from downstream analysis if they failed to achieve a RPKM (reads per kilobase of exon length per million mapped reads) value of at least 0.5 in at least two libraries. Differential expression analysis was performed using Biconductor R package edgeR. A negative binomial generalized log-linear model was fitted to each gene, and likelihood ratio tests were performed to assess differential expression. Genes were called differentially expressed if they achieved a false discovery rate of 0.1 or less and also had at least 8 RPKMs in one or both of the two cell types being compared.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files including log2-RPKM values for genes in each sample
|
| |
|
| Submission date |
Apr 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Wei Shi |
| E-mail(s) |
[email protected]
|
| Organization name |
Olivia Newton John Cancer Research Institute
|
| Department |
Bioinformatics and Cancer Genomics
|
| Street address |
Level 5, ONJ Cancer Centre, 145 Studley Rd
|
| City |
Heidelberg |
| State/province |
VIC |
| ZIP/Postal code |
3084 |
| Country |
Australia |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE68056 |
Transcriptional profiling of antigen-specific CD8 T cells from wildtype and mutant mice. |
|
| Relations |
| BioSample |
SAMN03492294 |
| SRA |
SRX1000335 |
