GSM8581585: LSG-SjD, scRNA-seq, GEX; Homo sapiens; RNA-Seq
Fresh tissue sample from surgically obtained SjD were preserved in Tissue Storage Solution (130-100-008, Miltenyi Biotec). Tissue sample was transferred into gentleMACS C tubes (130-093-237, Miltenyi Biotec) and cut into small pieces in RPMI-1640 with L-glutamine and phenol red (189-02025, FUJIFILM). Then the tissue was made into a cell suspension using the Multi Tissue Dissociation Kit I (130-110-201, Miltenyi Biotec) and the gentleMACS Octo Dissociator with Heaters (130-096-427, Miltenyi Biotec). The cell suspension was de-aggregated by 100 μm MACS SmartStrainer (130-098-463, Miltenyi Biotec) and transferred into a 5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap (352235, Falcon). After washing twice with EasySep buffer (20144, STEMCELL), erythrocytes were removed with Red Blood Cell Lysis Solution (130-094-183, Miltenyi Biotec). Following separation, cells were preserved in CELLBANKER 1 plus (11912, ZENOGEN PHARMA) and stored at -80°C for scRNA-seq. Positive selection of CD45+ cells was performed with the EasySep Human PE Positive Selection Kit II (17664, STEMCELL). Cells labelled with magnetic particles coated with anti-CD45+ antibody were separated using EasySep Magnet (18000, STEMCELL). The number and viability of separated CD45+ cells were assessed using a LUNA-FX7 automated cell counter (L70001, Logos Biosystems) and the cell concentration was adjusted to 10,000 single cells. These cells were then loaded into a 10x Genomics Chromium device according to the protocol of Chromium Single Cell 5' Reagent Kits (v2) as previously described.