Ii automated cell counter

The primary culture used for clonal analysis was also used for single-cell RNA-seq assay. Keratinocytes were detached with trypsin for 15–20 min in order to obtain a single-cell suspension and pelleted in culture medium. Cells were then suspended in 1× phosphate-buffered saline (PBS) with 0.04% BSA and filtered with 70 μM cell filter in order to discard any clamp or cell cluster. Cell suspension were then visualized and counted with trypan blue using a Countess™ II Automated Cell Counter to get a precise estimation of total number of cells and of cells concentration. Afterwards we loaded about 10,000 cells of each sample into one channel of the Chromium Chip B using the Single Cell reagent kit v3 (10X Genomic) for Gel bead Emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer’s protocol. Fifty nanograms of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq550 Illumina sequencing platform following the 10X Genomics instruction for read generation, reaching at least 50,000 reads as mean reads per cell.

Single-cell gel bead-in-emulsions (GEMs) were constructed using a Chromium Single Cell 5' Library and Gel Bead Kit following the manufacturer's instructions. Briefly, FACS-sorted cells were washed with 0.04% BSA PBS three times and resuspended to a concentration of 700 ~ 1200 cells/µL (viability ≥ 85%), as determined by a Countess™ II Automated Cell Counter. The cells in the samples that reached the standard were then captured in droplets. Single-cell GEMs consisted of single-cell and specific 16-nt 10× barcodes and 10-nt unique molecular identifiers (UMIs). Within the single-cell GEMs, the tagged cells were lysed, and the released mRNA was barcoded. Reverse transcription (RT) was performed in the GEMs. After the RT step, the emulsion was broken, and amplification of the 10× barcoded cDNA was completed. Amplified cDNA was then used for the construction of 5′ gene expression libraries. Each cDNA library was sequenced on a NovaSeq platform (Illumina) to generate 150-bp paired-end reads.