|
| Status |
Public on Aug 07, 2015 |
| Title |
S1 |
| Sample type |
RNA |
| |
|
| Source name |
dorsal pancreatic epithelia-e12.5-Sox9het
|
| Organism |
Mus musculus |
| Characteristics |
tissue: dorsal pancreatic epithelia
age: Embryonic day 12
genotype/variation: Sox9het
|
| Growth protocol |
All animal experiments described herein were approved by the University of California, Irvine and San Diego Institutional Animal Care and Use Committees. For timed matings, noon on the day of vaginal plug appearance was designated e0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
E12.5 dorsal pancreatic epithelia were manually microdissected and stored in 50 μl of RLT lysis buffer (QIAGEN, Valencia, CA) in the –80°C freezer. Each individual RNA sample was collected from three dorsal pancreata (i.e. three embryos) as per the manufacturer's instructions. We prepared RNA samples in ~12 μl of DEPC-treated water. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Approximately 250 ng of total RNA was amplified and labeled with Cy3 using the Low RNA Input Linear Amp Kit PLUS, One-Color (Agilent Technologies, Santa Clara, CA). This labeling reaction produces 0.25 – 2.0 μg of Cy3-labeled cRNA (anti-sense), by first converting mRNA primed with an oligo (d)T-T7 primer into dsDNA with MMLV-RT and then amplifying the sample using T7 RNA Polymerase in the presence of Cy3-CTP.
|
| |
|
| Hybridization protocol |
1.65 μg of cRNA was fragmented and hybridized to the array for 17 hr. at 65°C then washed.
|
| Scan protocol |
Arrays were scanned with the AgilentTechnologies Scanner G2505B US23502332 using protocol version GE1-v5_95_Feb07
|
| Data processing |
Mean foreground intensities were obtained for each spot and imported into the mathematical software package “R”, which is used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis. In outline, the Cy3 (green) intensities were not background corrected (this has been shown to only introduce noise), and corrected for the scanner offset (40 was subtracted for each intensity). The dataset was filtered to remove positive control elements and any elements that had been flagged as bad. Using the negative controls on the arrays, the background threshold was determined and all values less than this value were set to the threshold value. Finally, the data was normalized using the Limma Quantile Normalization package in “R” (Smyth 2004, Bolstad et al., 2003).
|
| |
|
| Submission date |
Oct 03, 2014 |
| Last update date |
Aug 08, 2015 |
| Contact name |
Maike Sander |
| E-mail(s) |
[email protected]
|
| Organization name |
UC San Diego
|
| Street address |
2880 Torrey Pines Scenic Dr.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE61948 |
Transcriptome and cistrome analysis reveals synergistic roles for Sox9 and Pdx1 in lineage allocation of foregut progenitor cells |
| GSE62023 |
Identification of Sox9/Pdx1-coregulated Genes During Pancreas Organogenesis |
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