|
| Status |
Public on Feb 10, 2017 |
| Title |
Embryonic mTEC Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Embryonic day 14.5 Fetal Thymus mTEC + 7days in Culture
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: E14.5 Fetal Thymus+ 7days in Culture
genotype: Wildtype
age: Embryonic day 14.5 + 7days in Culture
|
| Treatment protocol |
10-15 fetal thymi were dissected from E14.5 wildtype embryos and cultured for 7 days in AIM-V. For isolation of embryonic TEC, thymic lobes were dissected and washed once in PBS followed by an incubation of 15 min in PBS containing 0.25% trypsin and 0.02% EDTA and gentle mechanical dissociation to obtain cell suspensions. For isolation of adult TEC, thymic lobes were dissected and cut into small pieces (~10 mm × 10 mm), excess thymocytes removed by sequential washes in HBSS containing FCS 10% (v/v) and incubated for 30 min at 37 °C in HBSS containing FCS, 3.3 mg/ml Liberase and 0.5 mg/ml DNaseI (Roche Diagnostics UK). After incubation, cells suspensions were prepared by gentle mechanical dissociation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the PicoPure RNA Isolation Kit (ThermoFisher, Cat. KIT0202) performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Up to 5.5g of total RNA was fragmented and labelled using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay as outlined in the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
Hybridisation to Affymetrix Mouse Gene 2.0 ST arrays was performed for 16 hours at 45C with constant rotation. Samples were hybridised according to the Affymetrix specifications: hybridisation cocktails were heated at 99C for 5 minutes and then at 45C for another 5 minutes. Samples were centrifuged at max speed for 1 minute and 200ul was transferred to each array. Hybridisation was carried out for 16 hours at 45C and 60 rpm. Fluidics washing was FS450_0001.
|
| Scan protocol |
Arrays were scanned with an Affymetrix Gene ChIP Scanner 3000 7G
|
| Description |
Gene expression data from E14.5 Fetal Thymus mTEC 7 days in culture
|
| Data processing |
Data was normalised using rma function from the oligo package in R
|
| |
|
| Submission date |
May 13, 2016 |
| Last update date |
Feb 10, 2017 |
| Contact name |
Tessa Crompton |
| Organization name |
UCL
|
| Department |
Immunobiology
|
| Street address |
30 Guilford Street
|
| City |
London |
| ZIP/Postal code |
WC1N 1EH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE81433 |
Gene Expression Data from Adult and Embryo Thymic Epithelial Cells (TEC) |
|
