Gene expression profiling of mouse embryonic stem cells with single-cell RNA sequencing

Differences in gene-expression profiles between individual cells can give rise to distinct cell fate decisions. Yet how localisation on a micropattern impacts initial changes in mRNA, protein, and phosphoprotein abundance remains unclear. To identify the effect of cellular position on gene expression, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridization (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins, and phosphoproteins in single cells. We studied 67 (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns. To validate the detection of the mRNA targets quantified with ARTseq-FISH, we utilized single-cell RNA sequencing to profile a comparable population of mouse embryonic stem cells. Mouse embryonic stem cells (mESCs) were seeded and growth in serum/LIF conditions. 30 hours post-seeding, cells were detached from the culture surface and frozen in preparation for single-cell RNA sequencing. Single-cell RNA sequencing was performed from a freshly thawed sample, through single-cell barcoding (10x Genomics Chromium) and sequencing (NextSeq 2000; Single Cell 3'v3 chemistry).