|
| Status |
Public on Dec 29, 2017 |
| Title |
iNKT 4h after Transwell-separated co-culture with aGC-loaded NHBE epithelial cells, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
iNKT cells seprated by a transwell insert in co-culture system with glycolipid-loaded airway epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Invariant Natural Killer T lymphocytes
|
| Treatment protocol |
Human iNKT cells (non-adherent) were carefully aspirated from NHBE (adherent) epithelial cells after 4h co-culture, or used at 0h as untouched cells. Total RNA was isolated by RNeasy Mini Kit (Qiagen).
|
| Growth protocol |
Human iNKT cells were grown in RPMI-1640 medium supplemented with 10% human serum and 1% Penicillin/Streptomycin at 37C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction with Rneasy Mini Kit was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA (generated from cRNA, and after fragmentation) were prepared according to the standard Affymetrix protocol from 100-300 ng total RNA (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay, Affymetrix; http://media.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf).
|
| |
|
| Hybridization protocol |
Following fragmentation, cDNA molecules were labeled with biotin using uracil DNA glycosylase (UDG), and then hybridized for 16 hr at 45C on Human Whole Transcript (WT) 1.0 Sense Target (ST) Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Fluorescent images on GeneChips were captured using gene Array scanner 3000 and GCOS 1.2 software (Affymetrix).
|
| Data processing |
The flourescent data were initially analyzed with GeneChip Operating Software. Then in R, data were normalised using RMA algorithm and scripts were written to plot data in 3D. Gene-set enrichment analysis (GSEA) - http://www.broadinstitute.org/gsea/index.jsp, and Database for Annotation, Visualization and Integrated Discovery (DAVID) - http://david.abcc.ncifcrf.gov/, were also used for data analysis.
|
| |
|
| Submission date |
Feb 21, 2012 |
| Last update date |
Dec 29, 2017 |
| Contact name |
Kambez Hajipouran Benam |
| Organization name |
Wyss Insstitute, Harvard Medical School
|
| Street address |
Center for Life Science Boston, 3 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE35987 |
Expression data from human iNKT cells with or without interaction with human airway epithelium |
|
