Fluidigm c1 chips

Frozen viable pleural effusion or ascites vials were thawed and CD45+ white blood cells were depleted using Miltenyi’s QuadroMACS. Individual cells were captured using Fluidigm C1 chips (10–17 μm cell diameter); whole-genome amplification was performed per manufacturer’s instructions. Targeted amplification of mutation-containing regions was performed using Fluidigm Access Array and BioMark instruments per manufacturer’s instructions. Libraries were sequenced on Illumina MiSeq. Reads were aligned to hg19 with BWA MEM v0.7.8. Variants were called using MuTect v1.1.4. Co-occurrence of mutations (at least 1 mutant read) in individual cells was evaluated using Fisher’s exact test. Single cells with more than one subclone-defining mutation are shown.

For the scRNA-seq, epidermal cells were isolated from the back skin of 8-week-old Ivl-traced mice as described previously¹⁹ . Cells were stained for 1 h with CD49f (Itga6)-AlexaFluor 647 (1:50; BD Biosciences; catalogue number 551129), Sca1(Ly6a)-PeCy7 (1:50; BD Biosciences; catalogue number 558162) and Cd34-FITC (1:50; BD Pharmingen catalogue number 553733) and Sytox blue (1:1,000; Life Technologies catalogue number S34857) was added just before (2 min before) FACS sorting on FACSAria III machine with BD FACSDiva 8.0.1 software (BD Biosciences). Tomato-traced and non-traced live cells gated for ITGA6⁺/SCA1⁺/CD34⁻ were collected in 400 μl Defined Keratinocyte serum-free medium (Thermo Fisher, catalogue number 10744019) with 1% DNaseI (Stem Cell Technologies, catalogue number 07900), loaded into Fluidigm C1 chips (Fluidigm) for library preparation and sequenced as described previously¹⁹ .