GSM6481637: normal-1; Homo sapiens; RNA-Seq
Six tissue samples (normal = 1, NEOLP = 3, EOLP = 2) were washed by D-PBS twice and cut into fragments with a diameter of about 1-2mm, Infiltrating in the mixture of Whole Skin Dissociation Kit human (Miltenyi). The mixture was digested in water bath at 37 ℃ for 2.5 hours and dissociated by gentleMACSTM Octo Dissociator for 15 seconds every 0.5-1hours. Next, the mixture was filtered by 70 μm MACS Smartstrainers (Miltenyi), added 1ml DMEM to terminate digestion, and 4 ℃ centrifuged at 400rcf for 15 minutes. The cell precipitates were again subjected to lysis of red blood cells by Red Blood Cell Lysis Buffer (Abcam) and removal of dead cells by Easysep Dead Cell Removal (Annexin V) Kit (STEMCELL). Finally, single cell suspension was resuspended by D-PBS and counted through trypan blue. The method of library establishment was as previous described (Xu et al., 2020). In brief, the single cell suspension was combined with barcoded beads to produce single-cell Gel Beads-in-Emulsion (GEMs) on the 10×Genomics platform. GEMs were dissolved, cells were lysed to release mRNA, and cDNAs were amplified. Library was constructed by the 10× Genomics Chromium Single Cell Kit (v3 chemistry) and sequenced on an Illumina NovaSeq 6000 System.