|
| Status |
Public on Mar 26, 2014 |
| Title |
Irradiated Worm Pharynx Wound Plug 6 hrs Post Amputation Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Irradiated Worm Tissue Plug 6 hrs
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
time: 6 hr
radiation: 10k rad
replicate: 2
|
| Treatment protocol |
Irradiated animals were exposed to 10,000 rads on a GammaCell 40 Exactor irradiator. Planarian water was replaced with 100mM sodium azide (diluted in Montjuïc water). After 5-7 minutes, the pharynx was visibly extended out of the body. Vigorous pipetting often dislodged the pharynx from the body; if necessary, fine serrated forceps were used to remove the pharynx, followed by several washes in Montjuïc.
|
| Growth protocol |
Schmidtea mediterranea asexual clonal line CIW4 was maintained and used as previously described (Newmark and Sánchez Alvarado, 2000).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At specified times after amputation, plugs were extracted using 1mm microcapillary pipets (FHC, catalog # 30-30-0), and transferred directly into Trizol (Life Technologies) using a mouth pipet. For each replicate, 25 plugs were homogenized together, and then chloroform-extracted. The pellet was then precipitated with isopropanol, washed, and resuspended in water. RNA was then passed over an RNEasy column and DNasetreated.
|
| Label |
Cy5
|
| Label protocol |
Starting with 100 ng total RNA, amplification and labeling with Cy3 or Cy5 was performed using the Low Input Quick Amp Labeling Kit Two-Color from Agilent Technologies (#5190-2306).
|
| |
|
| Channel 2 |
| Source name |
Irradiated Worm Tissue Plug 0 hrs
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
time: 0 hr
radiation: 10k rad
replicate: 2
|
| Treatment protocol |
Irradiated animals were exposed to 10,000 rads on a GammaCell 40 Exactor irradiator. Planarian water was replaced with 100mM sodium azide (diluted in Montjuïc water). After 5-7 minutes, the pharynx was visibly extended out of the body. Vigorous pipetting often dislodged the pharynx from the body; if necessary, fine serrated forceps were used to remove the pharynx, followed by several washes in Montjuïc.
|
| Growth protocol |
Schmidtea mediterranea asexual clonal line CIW4 was maintained and used as previously described (Newmark and Sánchez Alvarado, 2000).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At specified times after amputation, plugs were extracted using 1mm microcapillary pipets (FHC, catalog # 30-30-0), and transferred directly into Trizol (Life Technologies) using a mouth pipet. For each replicate, 25 plugs were homogenized together, and then chloroform-extracted. The pellet was then precipitated with isopropanol, washed, and resuspended in water. RNA was then passed over an RNEasy column and DNasetreated.
|
| Label |
Cy3
|
| Label protocol |
Starting with 100 ng total RNA, amplification and labeling with Cy3 or Cy5 was performed using the Low Input Quick Amp Labeling Kit Two-Color from Agilent Technologies (#5190-2306).
|
| |
|
| |
| Hybridization protocol |
Custom Agilent 4x44k arrays with design id: 033226 were hybridized using oligoarray control targets and Agilent hybridization buffers according to the manufacturer protocols.
|
| Scan protocol |
Arrays were scanned using an Agilent G2505C scanner and quantified with Agilent Feature Extraction software version 10.5 using protocol GE2_105_Dec08.
|
| Data processing |
Data was analyzed in the R environment using the Limma library (Smyth, 2004) for loess normalization and calculation of p-values between treatments.
|
| |
|
| Submission date |
Mar 25, 2014 |
| Last update date |
Mar 26, 2014 |
| Contact name |
Chris W Seidel |
| E-mail(s) |
seidel@phageT4.org
|
| Phone |
816 926 9054
|
| Organization name |
Stowers Institute
|
| Department |
Genomics
|
| Lab |
Seidel
|
| Street address |
1000 E 50th St
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL18465 |
| Series (1) |
| GSE56181 |
Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria |
|
