|
| Status |
Public on Sep 21, 2020 |
| Title |
Ectoderm sample 2 from chick treated with 40-1A ctrl cells |
| Sample type |
RNA |
| |
|
| Source name |
Frontonasal ectodermal zone ectoderm
|
| Organism |
Gallus gallus |
| Characteristics |
tissue: Frontonasal ectodermal zone ectoderm
treatment: Injected with 40-1A control cells
|
| Treatment protocol |
Inhibition of neural SHH signaling was achieved by injecting hybridoma cells expressing either immuno‐neutralizing anti‐SHH antibody (5E1) or anti‐β‐galactosidase antibody control cells (40-1A) into the neural tube at Stage 10 (HH10). SHH-N treatment was performed using affi-Gel Blue beads (50 – 100 mesh, 200 – 250 diameter) soaked in recombinant SHH-N protein (400 ng/ml in PBS with 0.1% bovine serum albumin (BSA)) or 0.1% BSA (control) at 37° C for one hour. Forty-eight hours after SHH signal inhibition, SHH-soaked beads were placed between the facial ectoderm and the forebrain neuroectoderm. Embryos were collected at HH22 for genomic analysis.
|
| Growth protocol |
Fertilized chicken eggs (Gallus gallus, Rhode Island Red, Petaluma Farms, CA) were incubated at 39° C in humidified conditions and prepared for surgical manipulation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Agilent manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
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 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 on the Agilent scanner immediately after washing.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.1 (Agilent) using default parameters)
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| |
|
| Submission date |
Jan 29, 2020 |
| Last update date |
Sep 21, 2020 |
| Contact name |
Andrea J Barczak |
| Organization name |
UCSF Sandler Center Functional Genomics Core Facility
|
| Street address |
1550 4th St Rock Hall 545
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL28095 |
| Series (1) |
| GSE144499 |
miR-199 Family Regulates Sonic Hedgehog Expression During Craniofacial Development |
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