|
| Status |
Public on Oct 01, 2016 |
| Title |
Naïve_RNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
CD8 T cell
|
| Organism |
Mus musculus |
| Characteristics |
cell stage: Naïve
strain: C57BL/6
|
| Treatment protocol |
Naive CD45.2+ P14 TCR-Tg CD8+ T cells were isolated from the lymph nodes and adoptively transferred into CD45.1+ naïve recipient mice at 10,000 cells per mouse. One day later, the recipient mice were i.p. infected with 2×105 PFU of LCMV-Armstrong.
|
| Growth protocol |
C57BL/6 and B6.SJL mice were from the Jackson Laboratory. All mouse experiments were performed under protocols approved by the Institutional Animal Use and Care Committee of the University of Iowa.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
On day 8 post-infection (p.i.), CD45.2+CD8+KLRG1+ IL-7Ra– cells were sort-purified as terminally differentiated effector CD8+ T cells. On ≥ day 60 p.i., CD45.2+CD8+CD62L+ cells were purified as central memory CD8+ T cells. From the lymph nodes of uninfected P14 TCR-Tg mice, CD62L+CD44lo-med CD8+ cells were isolated as naïve CD8+ T cells. The cell sorting was performed on on FACSAria (BD Biosciences).
Total RNA was isolated from sorted CD8+ T cells. RNA Integrity was assessed using RNA 6000 Pico Kit (5067-1513) for Bioanalyzer 2100 (Agilent). mRNA was isolated from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). PolyA-selected mRNA was fragmented to an average size of 300 nt, reverse transcribed to generate double-stranded cDNA, and converted to a paired end library using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) according to manufacturer's instructions including the optional double size selection procedure using Agencourt AMPure XP beads. Prepared libraries were quantified with dsDNA HS Kit (Q32851) for Qubit and the size distribution was assessed using High Sensitivity DNA Kit (5067-462) for Bioanalyzer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basecalls performed using Illumina Casava version 1.8
RNA-Seq reads were mapped to mouse genome (mm9) using Tophat. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses.
Transcripts were assembled using Cufflinks using mapped fragments outputted by Tophat. Ensembl (release 65) was used as the source of annotated genes and transcript isoforms.
Mapped reads were assigned to the annotated genes/transcripts using the Rsubread package (v1.16.1). Differential expressed genes/transcripts between two cell types were identified using the EdgeR method (v3.8.6) and FDR < 0.05 and fold change > 2.
Genome_build: mm9
|
| |
|
| Submission date |
Dec 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Bing He |
| Organization name |
Children's Hospital of Philadelphia
|
| Lab |
Kai Tan
|
| Street address |
3501 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19074 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE76030 |
Dynamic organization and activation of enhancers and super-enhancers dictate effector and memory CD8+ T cell responses (RNA-Seq) |
| GSE76031 |
Dynamic organization and activation of enhancers and super-enhancers dictate effector and memory CD8+ T cell responses |
|
| Relations |
| BioSample |
SAMN04338386 |
| SRA |
SRX1482919 |
