GSM9031941: Human, skin, pustular, spatial, rep1; Homo sapiens; OTHER
The collected tissue samples were immediately processed for cell dissociation. After washing with PBS (Gibco, 10010023), adipose tissue was carefully removed. The tissue was finely minced using a scalpel and scissors, then incubated in Liberase TL solution (0.25 mg/mL; Miltenyi Biotec, 05401020001) at 37 °C for 40 minutes, with gentle vortexing or tapping every 5 minutes. Following enzymatic digestion, the sample was resuspended by pipetting 10 times using a wide-bore pipette tip. The resulting suspension was filtered through a 70 μm cell strainer and subsequently rinsed with PBS. Cells were pelleted by centrifugation at 300 ×  g for 5 minutes at 4 °C, resuspended in 1 mL of freezing medium (Cellbanker 1; Nippon Zenyaku Kogyo, BLC-1), and cryopreserved in liquid nitrogen. For the sequencing step, cryopreserved samples were thawed in a 37 °C water bath. Once only small ice crystals remained, samples were removed and 1 mL of pre-warmed culture medium (RPMI; HyClone, SH30027.01) supplemented with 10% FBS (Thermo Fisher, A3160401) was added. Cells were gently resuspended using a wide-bore pipette tip and transferred to a 15 mL Falcon tube containing 9 mL of additional warm culture medium. After centrifugation at 300 × g for 5 minutes at 20 °C, the supernatant was removed and the cell pellet was gently resuspended in 1 mL of warm culture medium. An initial cell count was performed, followed by filtration through a 40 μm cell strainer. After an additional centrifugation and resuspension step, the final cell suspension was prepared in PBS containing 0.04% BSA (Gibco, 15260037). A final cell count was recorded prior to library generation. Spatial sequencing libraries were generated using the Visium HD Human Transcriptome Kit (10x Genomics, 1000675) according to the manufacturer's instructions at Macrogen Inc. (Seoul, Korea). FFPE tissue blocks with DV200 values greater than 30% were selected for library preparation, and 5 μm sections were used. Libraries were sequence on a Novaseq system (Illumina).