Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: human sample collection: skin samples were obtained through punch biopsy (4 mm), 10 cm distal to the elbow and placed in saline containing tubes on ice and then transferred to cold FACS buffer (EDTA pH8.0 2mM, BSA 0.5% in PBS). Whole blood samples were collected at the time of skin biopsy, were placed in EDTA-containing tubes (Beckton Dickenson) on ice and diluted 1:1 with ice cold FACS buffer. Skin and blood samples immediately transported to the lab. Skin cell suspension and blood mononuclear cells were stained with the following antibodies: CD45 (PerCp/Cyanine, 3040285.5, Biolegend), CD90 (PE 328144, Biolegend), LGR5 (PE, 373803, Biolegend), CD55 (FITC, 311306, Biolegend). All FACS antibodies were used with a 1:100 dilution. Samples were filtered through a 40μm strainer before commencing sorting. Single cell sorting was performed using FACSAria Fusion cell sorter (BD Biosciences, San Jose, CA). After doublets exclusion, isolated live cells were single-cell sorted into 384-well cell capture plates containing 2μL of lysis solution and barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq. Four empty wells were kept in each 384-well plate as a no-cell control for data analysis. Immediately after sorting, each plate was spun down to ensure cell immersion into the lysis solution, snap frozen on dry ice, and stored at –80°C until processed. Cells were analyzed using BD FACSDiva software (BD Bioscience) and FlowJo software (FlowJo, LLC). Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014}