For assessing gene expression in Dlxi1/2b::eGFP+ cells in hSS and in hCS of fused hSS-hCS, we used a single-cell RNA-Seq assay adapted from the Smart-seq-2 protocol reported by Picelli et al51. In short, hSS and hCS that had been fused for ~15 days were separated with a scalpel blade and dissociated independently as described. Single-cells were isolated by FACS into a 96-well PCR plate containing 5 μl of lysis buffer containing 0.04% Triton X-100 (10%, Sigma BioUltra), 0.1 μl recombinant RNase inhibitor (TaKaRa), 1 μl Oligo-dT30VN (10 μM), 1 μl of 10 mM dNTP mix (Fermentas) and nuclease-free H20 for a final volume of 5 μl. A known number of internal RNA control (ERCC) was added to the lysis master mix to estimate the technical variability between the wells of the same plate and between plates. Reverse transcription and PCR amplification were performed using the parameters described by Picelli et al51. The quality of the cDNA library was checked using a High-Sensitivity DNA chip (Agilent Bioanalyzer). Libraries were prepared using the Nextera XT library prep kit (96 index primers, Illumina).
