title: Illumina HiSeq 4000 paired end sequencing; Fine mapping of cell states across anatomical sites in healthy human skin and basal cell carcinoma - scRNAsequencing
design: Fine mapping of cell states across anatomical sites in healthy human skin and basal cell carcinoma - scRNAsequencing
library_construction_protocol: Whole skin biopsies were kept on ice immediately after resection from facial skin for no longer than 2hrs before processing. The skin samples were either directly processed for dissociation into cell suspension or kept in MACS Tissue Storage Solution (Miltenyi Biotec, Surrey, U.K, cat. no. 130-100-008) overnight. The enzymatic and mechanical dissociation of whole skin biopsies was performed using the Whole Skin Dissociation kit for human material (Miltenyi Biotec, Surrey, U.K, cat. no. 130-101-540). Larger specimens were reduced in size by pre-cutting into several small pieces prior to enzymatic digestion and the quantity of enzymes used was adjusted according to the size of the sample. All biopsies were incubated with the enzyme solution for 3 hours in a shaking water bath at 37°C. Cell suspensions were then filtered through 70-µm cell strainer (Falcon) and centrifuged at 1500rpm for 10 minutes at 4°C. The collected cells were then frozen in 10%DMSO in Fetal Bovine Serum (FBS) and stored in liquid nitrogen. For library preparation, cells were thawed and resuspended in PBS with 2% FBS and DAPI (0.05mg/ml). Dead cells were removed through flow cytometry of the final cell suspension. Immediately after flow cytometry, ~20,000 to 100,000 live single cells were loaded onto a Chromium chip and libraries were prepared through droplet encapsulation on the Chromium controller (10X Genomics; Pleasanton,  CA,  USA) using the Single Cell 3' reagent kits following the manufacturer's protocol. Library quantification was performed using the Qubit dsDNA HS Assay Kit (Life Technologies, Waltham, MA, USA), and cDNA integrity was assessed using D1000 ScreenTapes (Agilent Technologies).