RNAScope analysis of human fetal tooth germ
Cite this dataset
Regier, Mary (2023). RNAScope analysis of human fetal tooth germ [Dataset]. Dryad. https://doi.org/10.5061/dryad.qnk98sfkk
Abstract
This data was collected to verify the spatial localization of human fetal tooth germ cell types identified via sn-RNA-seq. Using this and other data, we have proposed a more precise developmental timeline and trajectory for human tooth development.
Methods
Description of methods used for collection/generation of data: We used a RNAscope™ HiPlex12 Reagent Kit (488, 550, 650, 750) v2 Standard Assay (Advanced Cell Diagnostics, Inc.) and probes against 13 transcripts VWDE, SALL1, FGF4, IGFBP5, FGF10, PRRX1, FBN2, ENAM, PCDH7, SOX5, KRT5, and either DSPP or LGR6.
Probe/Channel Key:
- Round 1
- DAPI R1C1
- VWDE R1C2
- SALL1 R1C3
- FGF4 R1C4
- IGFBP5 R1C5
- Round 2
- DAPI R2C1
- DSPP R2C2
- LGR6 R2C2
- FGF10 R2C3
- PRRX1 R2C4
- FBN2 R2C5
- Round 3
- DAPI R3C1
- PCDH7 R3C3
- SOX5 R3C4
- KRT5 R3C5
Fresh frozen human fetal tooth germ tissue sections were stained according to the ACD protocol: https://acdbio.com/sites/default/files/UM%20324409%20RNAscope%20HiPlex%20v2%20User%20Manual%20%28488%2C%20550%2C%20650%2C750%29.pdf and imaged using a Nikon Ti2 with an Aura light engine and BrightLine Sedat filter set optimized for DAPI, FITC, TRITC, Cy5 & Cy7 (LED-DA/FI/TR/Cy5/Cy7-5X5M-A-000) for samples 1-4 or using a Yokogawa CSU-X1 spinning disk confocal microscope with a Celesta light engine and ORCA-Fusion scientific CMOS camera for sample 5.
Methods for processing the data: DAPI channel images for imaging rounds two and three were aligned to the DAPI image for imaging round one using the BigDataViewer > BigWarp plugin in Fiji. The resultant landmark tables were used in a custom .groovy script to align the DAPI, FITC, Cy3, Cy5, and Cy7 images from the three rounds of imaging in Fiji. Aligned images were background corrected and converted to a hyperstack in Fiji.
Describe any quality-assurance procedures performed on the data: Thresholding determined using positive and negative control sections
Usage notes
Instrument- or software-specific information needed to interpret the data: Fiji (ImageJ2 v2.3.0).
Funding
Brotman-Baty Institute
Dr. Douglass L. Mourell Research Fund
John H. Tietze Foundation Trust
Washington Research Foundation, ARCS
National Institute of Dental and Craniofacial Research, Award: 1P01GM081619
National Institute of Dental and Craniofacial Research, Award: R01GM097372
National Institute of Dental and Craniofacial Research, Award: R01GM97372-03S1
National Institute of Dental and Craniofacial Research, Award: R01GM083867
National Institute of Dental and Craniofacial Research, Award: U01HL099997
National Institute of Dental and Craniofacial Research, Award: UO1HL099993
National Institute of Dental and Craniofacial Research, Award: 5R24HD000836
National Institute of Dental and Craniofacial Research, Award: T90DE021984
National Institute of Dental and Craniofacial Research, Award: 1R01DE033016-01