Single cell RNA sequencing identifies CXADR as a fate determinant of the placental exchange surface
Tissue formation depends on adequate progenitor cell pool expansion followed by appropriate lineage specification events. Failures in these steps lead to anatomical defects that can have detrimental consequences, in particular when they occur during in utero development. Here, we subjected mouse trophoblast stem cells (TSCs) to targeted differentiation protocols to recapitulate the earliest steps in placenta formation, and followed the early lineage differentiation events by single cell sequencing approaches.
1. In vitro differentiation of mouse TSCs was induced by inhibition of MEK with PD0325901 (2µM Final) (Inhibit dataset) or withdrawal of the stemness-maintaining components FGF4 and embryonic fibroblast conditioned medium (Remove dataset). Samples were collected at t0 (stem cell state, n=2), 1h (n=4 Inhibit and Remove), 4h (n=4 Inhibit and n=3 Remove), 24h (n=4 Inhibit and n=2 Remove), 36h (n=4 Inhibit and Remove) and 48h (n=4 Inhibit and Remove). Cells were dissociated and single cell barcoded transcriptome libraries were generated using Drop-Seq. Libraries were sequenced across multiple lanes of Illumina Nextseq, HiSeq2500 and HiSeq4000 flow cells in 6 batches. We have added Seurat objects of the Inhibit, Remove, t0 and t24 datasets, Monocle gene module results for the Inhibit and Remove datasets and SCENIC regulon results for the Inhibit and Remove dataset.
2. For the scRNAseq dataset including CXADR KO and WT mTSCs the experiment was repeated and samples at 0h and 24h were collected with 3 independed clones per condition. The cells were processed with the Parse Evercode WT v3 kit according to the manufacturer's instructions and libraries were sequenced on 2 lanes of NovaSeq X 25B flowcell. Sequencing data was analysed on the Trailmaker platfrom (Parse Biosciences, 2024) on 28/08/2024. Cell proportion analysis was performed using propeller from the package speckle. We have added Seurat objects for the Inhibit and Remove condition, data filtering and analysis settings from the Trailmaker platform, DEG tables for clusters at res. 0.7, input tables for the propeller analysis. We have also added a sample loading table needed to re-run the Parse pipeline.
3. We also include a table of source data for all bar charts/graphs in the manuscript.
