GSM4824577: K82_t2; Homo sapiens; RNA-Seq
Keratinocytes were detached with trypsin for 15-20 minutes in order to obtain a single-cell suspension and pelleted in culture medium. Cells were then suspended in 1X PBS with 0.04% BSA and filtered with 70uM cell-filter in order to discard any clamp or cell-cluster. Cell suspension were then visualized and counted with trypan blue using 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 3' 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. 50 ng 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 10xGenomics instruction for read generation, reaching at least 50000 reads as mean reads per cell.