Pathogenic Keratinocyte States and Fibroblast Niches Define the Tissue Microenvironment in Severe Hidradenitis Suppurativa
Pathogenic Keratinocyte States and Fibroblast Niches Define the Tissue Microenvironment in Severe Hidradenitis Suppurativa
Skin from HS surgery was kept in a cold environment immediately after resection and either directly processed for dissociation into cell suspensions or kept in MACS Tissue Storage Solution (Miltenyi Biotec, cat. no. 130-100-008) overnight. Large samples were cut into small pieces (0.5-1cm2) prior to further experimentation. Skin was enzymatically digested to separate epidermis from dermis with Dispase overnight at 4°C. The IFE was digested with a mixture of trypsin-EDTA and Versene for 15 minutes at 37oC. The dermis was dissociated following the Whole Skin Dissociation kit for human material (Miltenyi Biotec, cat. no. 130-101-540). Dermis samples were incubated with the enzyme solution for 3 hours in a shaking water bath at 37°C. Fetal bovine serum (FBS) was then added to the mix to terminate the process, and the cell suspensions were then filtered through 70-µm and 40µm cell strainers (Falcon). Cell suspensions were centrifuged at 1200rpm for 4 minutes at 4°C and supernatant was carefully aspirated. The collected cells were frozen in 10% DMSO in (FBS) and stored in liquid nitrogen tanks.  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). 