RNAScope analysis of human fetal tooth germ

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