Red cell lysis buffer

Spleen tissues were fragmented into small pieces and pressed against a 200-gauge steel mesh. Cell suspension was collected, and erythrocytes were lysed by red cell lysis buffer (Sangon Biotech Shanghai Co. Ltd., Shanghai, China). Cells were resuspended and adjusted to a concentration of 1 × 10⁶/ml in Dulbecco's Modified Eagle Medium (DMEM) (Sangon, China) containing 15% fetal bovine serum and 1% penicillin and streptomycin.
Skin tissues were cut into 0.5 cm × 0.5 cm pieces and soaked in 0.5% trypsin (Sigma-Aldrich, USA) at 37°C for 2 hr. After separating the dermis and epidermis, the dermis was shaken and digested with DMEM containing collagen enzyme IV (Sigma-Aldrich, USA) and deoxyribonucleic acid enzyme I (DNase I) (Thermo, USA) at 90 rpm for 1 hr. Then, cells were resuspended and adjusted to a concentration of 1 × 10⁶/ml.

Whole blood of 10 patients (IDs: 19010101, 19010102, 19010104, 190101010, 19010111, LXY, DTY, LC, OYZY, and GJX) and 10 healthy controls were first processed with Red cell lysis buffer (Sangon Biotech, Shanghai, China) and then treated with TRIzol (Invitrogen, Carlsbad, CA, USA) to extract total RNA. Reverse transcription was performed with HiScript First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) to obtain cDNAs. Then qPCR was performed on Bio-Rad CFX96 (Bio-Rad Laboratories Inc., Hercules, CA, USA) with AceQ qPCR SYBR Green Master Mix (without ROX) (Vazyme, Nanjing, China) according to the manufacturers’ protocols. ACTB gene was used as the reference, and the primer sequences are listed in Supplementary Table S1. Cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 25 s.