|
| Status |
Public on Dec 31, 2017 |
| Title |
mDC-educated OT-I CD8 T Cells 48h 1 |
| Sample type |
SRA |
| |
|
| Source name |
CD8 splenocytes (neg selection) from OT-I mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: OT-I
|
| Growth protocol |
OT-I cells were selected by negative magnetic selection of CD8 cells from whole spleen of an OT-I mouse. They were then co-cultured on a monolayer of flow cytometry-sorted lymphatic endothelial cells (LECs; podoplanin- and CD31- double-positive), which had been pre-pulsed with ovalbumin and washed prior to addition of OT-I cells at a ~10:1 T:LEC ratio. As a control, bone marrow-derived dendritic cells (DCs) were generated ia the Lutz protocol, and matured with LPS and pulsed with ovalbumin prior to addition of OT-I cells at the same ratio. Co-cultures proceeded for up to 3 days, and because LECs and DCs eventually adhere to the plates, non-adherent cells were removed and treated for sequencing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNeasy Plus kit to isolate total RNA, which was then submitted to EPFL Gene Expression Core Facility for library preparation and sequencing. Their standard protocol involves poly-A selection and RT-PCR for selection of mRNA, and library preparation using the Illumina TruSeq mRNA library prep kit.
Illumina TruSeq mRNA library prep kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
RNA isolated from OT-I cells with Qiagen Rneasy Plus kit, and poly-A selection with RT-PCR was used to select for mRNAs for creation of libraries for sequencing (Illumina TruSeq Stranded mRNA library prep kit).
|
| Data processing |
FastQC (Babraham Bioinformatics, Cambridge, UK) - Sequence quality and contamination check
Read mapping and counting with HTSStation platform, which uses Bowtie2 algorithm.
Differential expression analysis (pairwise between all 7 conditions) performed using Bioconductor Limma.R package.
Adjusted p-values calculated with Benjamini-Hochberg multiple tsting adjustment.
Genome_build: GRCm38 mm10
Supplementary_files_format_and_content: PRJNA347820_Genes_Counts&RPKMs.xls is a list of RPKMs and raw counts for each of the 21 samples, following gene calling. PRJNA347820_Transcripts_Counts&RPKMs.xls is an array of RPKMs and counts for detected transcripts. All other files (provided as .txt) contain adjusted p-values and Log-Fold Changes for the pairwise comparison indicated in the filename (n = 3 per condition), based on either gene calling or raw transcripts.
|
| |
|
| Submission date |
Oct 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shann S Yu |
| E-mail(s) |
shannyu@uchicago.edu
|
| Organization name |
University of Chicago
|
| Department |
PME-Immunoengineering Center
|
| Street address |
5640 S Ellis Ave, Eckhardt Research Center
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE88830 |
CD8 T cell phenotype following antigen education by lymphatic endothelial cells versus mature dendritic cells |
|
| Relations |
| BioSample |
SAMN05894985 |
| SRA |
SRX2239680 |
