Illumina HiSeq 4000 paired end sequencing; GSM2667058: AG1neg_1, Untreated single cell RNA-seq; Mus musculus; RNA-Seq

AG1neg_1, Untreated single cell RNA-seq

We performed single-cell mRNA-Seq on wild-type mouse keratinocytes co-cultured with keratinocytes in which beta-catenin was activated. We identified seven distinct cell states in cultures that had not been exposed to the beta-catenin stimulus. Using temporal single-cell analysis we reconstruct the cell fate changes induced by neighbor Wnt activation. Gene expression heterogeneity was reduced in neighboring cells and this effect was most dramatic for protein synthesis associated genes. The changes in gene expression were accompanied by a shift from a quiescent to a more proliferative stem cell state. By integrating imaging and reconstructed sequential gene expression changes during the state transition we identified transcription factors, including Smad4 and Bcl3, that were responsible for effecting the transition in a contact-dependent manner. Our data indicate that non cell autonomous Wnt/beta-catenin signaling decreases transcriptional heterogeneity and further our understanding of how epidermal Wnt signaling orchestrates regeneration and self-renewal. Overall design: Comparison of cells exposed to Wnt activated neighbors versus unactivated.

Epidermal Wnt signaling regulates transcriptome heterogeneity and proliferative fate in neighboring cells

Submitted by Gene Expression Omnibus on 22-DEC-2017

Wild type and K14ΔNβ-cateninER keratinocytes were cultured on 12 well plates in a ratio of 9:1 for a total of 200,000 cells per well and allowed to attach for 24 hours. Subsequently, cells were treated with 4-OHT (200nM) or DMSO as a control. After 24 hours of treatment cells were trypsinized and resuspended as a single cell suspension. Single keratinocytes were captured on a medium-sized (10-17μm) microfluidic chip (C1, Fluidigm). Cells were assessed for viability (LIVE/DEAD assay, Life Technologies) and C1 capture sites were imaged by phase contrast to determine empty and doublet capture sites. Cells were loaded onto the chip at a concentration of 300 cells μl-1. Doublet or non-viable cells were excluded from later analysis. Cell lysis, reverse transcription, and cDNA amplification were performed on the C1 Single-Cell Auto Prep IFC, as per the manufacturer’s instructions. For cDNA synthesis the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) was used. Single cell Illumina NGS libraries were constructed with Nextera XT DNA Sample Prep kit (Illumina). Sequencing was performed on Illumina HiSeq4000 (Illumina) using 100bp paired-end reads.

Epidermal Wnt signaling regulates transcriptome heterogeneity and proliferative fate in neighboring cells

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