|
| Status |
Public on Dec 31, 2016 |
| Title |
RNA-Seq_ALL02_OUT_TCM_IFNG |
| Sample type |
SRA |
| |
|
| Source name |
Blood PBMCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: IFNg secreting central memory CD4 Tcells - CD3+ CD4+ CD45RA- CCR7+ IFNG+
patient phenotype: Timothy Grass Allergic
seasonality: Blood draw out of Timothy Grass pollen season
|
| Treatment protocol |
4 million/ml PBMCs from 5 allergic and 6 non-allergic individuals were stimulated in vitro for 3 hours with 2.5 μg/ml of a Tymothy Grass Allergen immunodominant peptides pool (see paper).
|
| Growth protocol |
Cells were cultured for less than 18 hours in RPMI1640 supplemented with 5% human AB serum in 24 well plates at a density of 4x106/ml and incubated at 37˚C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
IFNγ or IL5 cytokine producing cells were captured using a cytokine secretion assay according to manufacturers’ instructions (Miltenyi Biotech, Germany). Briefly cells were harvested, washed and labeled with 20 μl of anti-IFNγ/CD45- or anti-IL5/CD45- antibody conjugates (Miltenyi Biotech). After cytokine capture, cells were stained with 20 μl/107 cells of PE-labeled detection antibody and co-stained with a combination of antibodies (Anti-CD3, -CD4, -CD45RA and –CCR7) to allow further separation of cells into effector memory (TEM; CD45RA-CCR7-), and central memory (TCM; CD45RA-CCR7+) by FACS sorting (FACS ARIA BD-Bioscience). Cells were sorted directly in 750ul Trizol LS. To maximize the cell lysis, samples were votexed every 5 minutes for 15 seconds.Total RNA was purified from the different sorted populations using a miRNAeasy kit (Qiagen, USA) as described as recommended by manufacturer. Purified total RNA (0.2–5ng) was amplified following the smart-seq2 protocol (Picelli et al. Nature methods, Vol10 (11), pp1096-98).
1 ng of amplified cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina, San Diego, USA).
Single-end 50bp length reads RNA-seq using the HiSeq2500 sequencer (Illumina, San Diego, USA)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
PolyAtail selected messenger RNA
Smart seq 2 cDNA amplification
|
| Data processing |
Illumina filtering
Alignment with TopHat
DUST scores were calculated with PRINSEQ Lite (v 0.20.3)
Low-complexity reads (DUST > 4) were removed from the BAM files
Generation of SAM files with SAMtools
Obtention of read counts with htseq-count using "union" option
Removing absent features (zero counts in all samples)
Raw counts were imported to R/Bioconductor package DESeq2 to identify differentially expressed genes among samples
DESeq2 normalizes counts by dividing each column of the count table (samples) by the size factor of this column. The size factor is calculated by dividing the samples by geometric means of the genes.
P-values for differential expression are calculated using binomial test for differences between the base means of two conditions.
These p-values are then adjusted for multiple test correction using Benjamini Hochberg algorithm to control the false discovery rate.
We considered genes differentially expressed between two groups of samples when the DESeq2 analysis resulted in an adjusted P-value of <0.05 and the fold-change in gene expression was 2-fold.
Genome_build: UCSC hg19
|
| |
|
| Submission date |
Sep 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Alessandro Sette |
| E-mail(s) |
alex@lji.org
|
| Organization name |
La Jolla Institute for Allergy and Immunology
|
| Street address |
9420 Athena Circle
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE70050 |
Lack of allergy to timothy grass pollen is not a passive phenomenon but associated with allergen specific modulation of immune reactivity |
|
| Relations |
| BioSample |
SAMN04028859 |
| SRA |
SRX1182552 |
