|
| Status |
Public on Mar 01, 2013 |
| Title |
Sample 3 Posterior +/+ |
| Sample type |
RNA |
| |
|
| Source name |
Posterior_wild type
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: C56B/6 and CD-1 mixed
strain background: wild type
age: E9.5
tissue: E9.5 embryos
microdissection: posterior second heart field
|
| Treatment protocol |
Posterior and anterior second heart field (SHF) tissues were isolated from each embryo and stored in RNAlater RNA stabilization reagent (QIAGEN) at 4 °C. Non-cardiac tissues were saved for genotyping. After genotyping, Shh -/- and Shh +/+ embryos were pooled, homogenized and RNA extracted.
|
| Growth protocol |
To get the Shh -/- mice, shh +/- female mice on mixed background were superovulated using 5 units each pregnant mare serum gonadotropin (PMS-G) and human chorionic gonadotropin (HCG) administered 48 hours apart. They were mated to Shh +/- male mice after HCG administration. Embryos were isolated at E9.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from pooled, homogenized SHF tissues using the QIAGEN RNeasy kit following the manufacturer's recommendations. The protocol includes an on-column DNase treatment. RNA was quantified using a Nanodrop ND-1000 spectrometer and quality was checked using an agilent bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cy3 labeled cRNA was created using the agilent quick-amp RNA labeling kit with RNA spike-in, followed by purification using a QIAGEN RNeasy kit. 0.5 ug was used for each sample, and the labeling was validated using a nanodrop spectrometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Technologies Scanner (G2505C US80900103) using one color scan setting for 4x44k array slides (Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
S3PosPls
slide barcode: 251486826533
US80900103_251486826533_S01_GE1_105_Dec08_1_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 014868_D_F_20090416) to obtain median background subtracted mean signal of the spot. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Feb 28, 2013 |
| Last update date |
Mar 01, 2013 |
| Contact name |
Xinan "Holly" Yang |
| E-mail(s) |
xyang2@uchicago.edu
|
| Phone |
773-702-5960
|
| Organization name |
universty of Chicago
|
| Street address |
KCBD5121, 900 E. 57th Str.
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE44754 |
Posterior Second Heart Field (pSHF) transcriptomes of wild-type and Shh mutant mouse embryos |
| GSE44756 |
Hedgehog Molecular Networks in the Second Heart Field |
|
