|
| Status |
Public on Nov 07, 2018 |
| Title |
Nelfb-cKO 24h-2c |
| Sample type |
RNA |
| |
|
| Source name |
cultured uterine stromal cells
|
| Organism |
Mus musculus |
| Characteristics |
nelf-b genotype: Cre+;flox/flox
tissue: uterine stromal cells
|
| Treatment protocol |
P4 (1 mM) and E (10 nM) for 24 hours
|
| Growth protocol |
Females were mated overnight with B6D2F1/J males, and uterine tissue was collected in the afternoon from 4 dpc females (morning of plug detection = 0.5 dpc), slit longitudinally,minced into 3- to 5-mm pieces, and washed several times with HBSS (MilliporeSigma). Uteri (2 to 3 each) were then placed in a 50-ml tubewith 5ml of 6 mg/ml dispase (Thermo Fisher Scientific) and 25 mg/ml pancreatin (MilliporeSigma) made in HBSS. Tissue was incubated for 60 min on ice, followed by 60 min at room temperature and then 10 min at 37°C.HBSS (30ml)with 10%FBS was added to each tube. Tissue was washed 2 times with 10 ml HBSS and moved to a 50-ml tube containing 0.5 mg/ml collagenase (MilliporeSigma) and incubated for 45min at 37°C.Tubes were vortexed until turbid. HBSS (30ml) + 10% FBS was added, and the suspension passed through a 70-mMfilter (MACS Smart Strainer;Miltenyi Biotec,Auburn, CA,USA). Cellswere collected by centrifuging at 450 g for 10 min and washed twice in 10 ml HBSS, then resuspended in DMEM-F12 (Thermo Fisher Scientific) containing 10% heat-inactivated fetal calf serum. Cells (400,000) in 3ml of media were seeded per well of a 6-well plate and allowedto attach for 1 to 2 h.Wellswere thenwashed several timeswithHBSS to remove nonadherent cells, and freshDMEMF12 containing 2%stripped fetal calf serumsupplementedwith P (1 mM) and E (10 nM) (PE, decidual stimulus)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
cells were collected with Trizol for RNA isolation
|
| Label |
biotin
|
| Label protocol |
Thirty five nanograms (35 ng) of total RNA were amplified as directed in the WT-Ovation Pico RNA Amplification System (Nugen, San Carlos, CA) protocol, sense-strand cDNA target was made using the Nugen Encore Exon Module, and after fragmentation the product was labelled with the Nugen Encore Biotin biotin module.
|
| |
|
| Hybridization protocol |
Five micrograms (5.0 μg) of amplified biotin-cDNAs were fragmented and hybridized to each array for 18 hours at 45°C in a rotating hybridization oven. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit and user manual following protocol FS450-0004.
|
| Scan protocol |
Arrays were scanned in an Affymetrix Scanner 3000.
|
| Description |
NELF-B UtcKO uterine stroma cells cultured for 24 hours with estradiol and progesterone to induce decidualization
|
| Data processing |
Data was obtained using the GeneChip® Command Console software.
|
| |
|
| Submission date |
Nov 07, 2018 |
| Last update date |
Nov 07, 2018 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL20775 |
| Series (1) |
| GSE122262 |
Transcriptional profiles of decidualized cultured WT vs NELF-B cKO Uterine stromal cells |
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