Transcriptional Profiling of Limb Progenitor Cells

Sox9⁺ cells were isolated from digits of E13.5 Sox9-GFP embryos (Nakamura et al., 2011 (link)). Lgr5⁺ and surrounding non-Lgr5 (Lgr5⁻) cells were isolated from forelimb digit interzones of E14.5 Lgr5^GFP/+ mice (Figures S3A–S3C). Cells were released with a mixture of TrypLE Express (Gibco) and 0.1% DNase I for 20 min, filtered through a 40 μm cell strainer, and sorted by FACS using an Aria I flow cytometer (BD Biosciences). Total RNA was extracted using a mirVana miRNA Isolation Kit (Ambion) and cDNA libraries were constructed with 10 ng of RNA using a SMARTer Ultra Low Input Kit (v3, Illumina), and sequenced using the Illumina HiSeq 1500 platform (Center for Genomic Sciences, The University of Hong Kong). cDNA fragment sequences were aligned to mouse genome (mm10) using the HISAT program (Kim et al., 2015 (link)). FPKM values were generated for comparison (Figure S3D). Genes with FPKM ≥ 5 were considered as expressing genes. Pathway and expression analyses were performed with DAVID (Huang da et al., 2009 (link)) and Eurexpress (Diez-Roux et al., 2011 (link)) databases, respectively. Datasets have been uploaded to the Gene Expression Omnibus for public access (GEO: GSE110281).