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Methods
The kumba (Ku) allele of Zic2 (described in 8,10) was maintained on two distinct backgrounds by continuous backcross to either C3H/HeH or C57BL6/N mice. For the production of staged embryos, 12 P.M. on the day of the appearance of the vaginal plug is designated 0.5 dpc.
Embryos were dissected at 14.5 dpc and a PTA-based staining protocol was used to provide soft tissue contrast. Briefly, embryos were rinsed 5 times with MilliQ water and then placed in 2.5% PTA within a decreasing ethanol series (70% : 7 days, 50% : 24 hrs, 30% 24 hrs) and finally 2.5% PTA in 4% PFA, with the solutions changed daily. μCT scanning was performed on a Caliper Quantum FX micro-CT using the maximal source voltage (90kV), source current (200uA) and a field-of-view of 20 (voxel resolution of 40μm). After scanning, the reconstructions (i.e. the scans) were saved as DICOM (.dcm) files with a resolution of 512x512x512 voxels.
Computing resources were provided by the National Computer Infrastructure within the Gadi computing cluster. Computing scripts (i.e. executable command lines of code - saved as .sh files) were submitted using the qsub system and the amount of cpus, memory and temporary storage was dependent on the script ran but can be seen commented with the first 10 lines of each .sh file.
DICOM files were converted into NRRD files using a custom script designed within the Arkell lab and then analysed via Lightweight Analysis of Morphological Abnormalities (LAMA; described in 11) but with a custom protocol suitable for scans obtained from the Quantum FX µCT machine.