GSM7468629: S100A8 HS2; Homo sapiens; OTHER
HS and healthy skin tissues were cut into small pieces and were digested with 2.5U/ml Dispase-II (Millipores Sigma, Cat#4942078001) in Hanks' Balanced Salt Solution (HBSS, Corning, Cat#21-022-CM) overnight at 4°C. After digestion, the epidermis was peeled off and minced. The minced epidermis was incubated with 0.05% trypsin-EDTA at 37°C for 40 mins for further digestion. The suspension was then filtered through a 40um cell strainer and spun down at 300g at 4°C for 5 mins.  Cells were then subjected to remove apoptotic and dead cells by using the Annexin Dead Cell Removal Kit (StemCell Technologies, 17899) and EasySep™ Magnet (StemCell Technologies, 18000). Enriched living cells were  were  for the application of CUT&RUN sequence. CUT&RUN experiments were carried out following the protocol of Epicypher CUTANA CUT&RUN kit (version 1.8). Briefly,  epidermal cells (1X10^4) were isolated with Wash Buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, 1X Roche cOmplete, protease inhibitor). The nuclei were captured with Concanavalin A (ConA) beads and incubated with 0.5 μg primary antibody for S100A8 (Abcam, cat#ab92331) in Antibody Buffer (Wash Buffer, 0.01% Digitonin, 2 mM EDTA) overnight at 4 °C. Then CUTANA pAG-MNase was added and incubated for 10 min at RT. CaCl2 was next added at a final concentration of 100 mM to activate the enzyme. The reaction was carried out for 2 hours at 4 °C and stopped by STOP buffer (340 mM NaCl, 20 mM EDTA, 4 nM EGTA, 50 μg/mL RNase A, and 50 μg/mL glycogen). Samples were incubated for 10 min at 37 °C in a thermocycler and quick spin down. DNA was purified using the CUANA DNA Purification Kit as the provided instructions. Purified CUT&RUN-enriched DNA was prepared sequencing libraries using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Cat# E7645s).