|
| Status |
Public on Aug 01, 2020 |
| Title |
[gene] LPS aCD3-aCD28 aIL10R diagnosis-CD subject-643 biological rep-11 |
| Sample type |
RNA |
| |
|
| Source name |
LPS_aCD3-aCD28_aIL10R
|
| Organism |
Homo sapiens |
| Characteristics |
stimulation: LPS_aCD3-aCD28_aIL10R
diagnosis: CD
Sex: M
batch: batch02
tissue: peripheral PBMC
|
| Treatment protocol |
For stimulation assays, 0.5×10^6 to 1×10^6 PBMC were cultured in 200 μl medium in duplicates in round bottom 96-well plates and exposed to ultrapure 200 ng/ml LPS (Enzo Life Sciences; Cat.# ALX-581-008), 200 ng/ml L18MDP (Invivogen) 10 μg/ml anti-IL-10R (Biolegend; clone: 3F9), and/or anti-CD3/anti-CD28 beads (Invitrogen) for 16 hours in complete RPMI with L-glutamine (Sigma) supplemented with 10 % human serum (Sigma; Cat.# H4522), non-essential amino acids (Gibco); 1 mM Sodium-Pyruvate (Gibco) and 100 U/ml penicillin and 10 μg/ml streptomycin (Sigma).
|
| Growth protocol |
Whole blood was collected into EDTA-containing tubes. PBMC were purified using Ficoll-Paque density gradient purification. The absolute number of cells was determined using a haemocytometer (Marienfeld).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultured cells, using the RNeasy Plus Mini Kit (Qiagen), according to the manufacturer’s protocol. The RNA yield was determined by quantifying the samples on an Agilent Bioanalyzer. Only samples with a 28S/18S ratio of 0.8 to 3.0 and a RIN Score of >= 6.0 passed QC metrics and were accepted for analysis.
|
| Label |
biotin
|
| Label protocol |
Minimum RNA input was 100ng with a concentration range of 10 ng/μl to 50 ng ng/μl. Label molecule was biotin. Amplification performed using standard protocol from Affymetrix WT Express Kit.
|
| |
|
| Hybridization protocol |
5 μg of biotin-labeled cDNA per HTA2 platform standard procedure
|
| Scan protocol |
Scanning performed with Affymetrix GeneChip station under default settings followed by CEL file derivation from Affymetrix GeneChip Command Console (AGCC)
|
| Description |
subject-ID_643
@52084400978709110317428272788109
|
| Data processing |
Background correction and quantile normalization of the gene expression data obtained from the Affymetrix Human Transcriptome Array 2.0 (HTA2) platform were performed by the RMA method while controlling for batch. Summarization was performed with RLM. Annotations were defined by the Affymetrix NetAffx(TM) NA35 release, which is based on the GRCh37 human reference genome. Differential expression at the gene-level was performed using the empirical Bayes method implemented in LIMMA{Ritchie:ef}controlling for donor in the model.
probe group file: HTA-2_0.r3.pgf
meta-probeset file: HTA-2_0.r3.Psrs.mps
|
| |
|
| Submission date |
Sep 18, 2019 |
| Last update date |
Aug 02, 2020 |
| Contact name |
Boyd Steere |
| E-mail(s) |
[email protected]
|
| Organization name |
Lilly
|
| Department |
Immunology
|
| Street address |
Lilly Corporate Center
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46285 |
| Country |
USA |
| |
|
| Platform ID |
GPL17586 |
| Series (2) |
| GSE137680 |
Gene expression analysis of PBMC's from IBD subjects stimulated by combinations of LPS, MDP, anti-CD3/anti-CD28 antibodies, and anti-IL10R antibodies. |
| GSE138009 |
Systems-level analysis of monocyte responses in inflammatory bowel disease identifies IL-10 and IL-1 cytokine networks that regulate IL-23 |
|
| Relations |
| Alternative to |
GSM4096041 (exon-level analysis) |
