GSM7106737
Once the patient was sedated and received analgesia, 10ml of 0.9% saline was injected into either the middle lobe of the right lung or the upper lobe of the left lung to obtain BALF. The BALF was passed through a 70μm nylon cell strainer (ThermoFisher Scientific, 22-363-548) to remove clumps and debris. The cells were then centrifuged at 300 g for 5 min at 4℃(LICHEN, LC-LX-LR55C), and the supernatant was discarded. The cells were re-suspended in PBS (Bioss, C7033) containing 0.04% BSA (Solarbio, s9020) and counted using a Countstar Rigel S2 instrument (Shanghai Ruiyu Biotechnology). We adjusted the cell suspension concentration to 300-600 living cells per microlite. Single-cell capturing and downstream library construction were performed using the Chromium Next GEM Single Cell 3'GEM Kit v3.1 (10x Genomics), following the manufacturer's protocol. In brief, a total of 70 μL of Master Mix and Cell Suspension (containing 6,000 cells), 50 μL of barcoded gel beads, and 45 μL of partitioning oil were loaded onto Chromium Chip K to generate single-cell gel bead-in-emulsion (GEM). Immediately following GEM generation, the gel beads were dissolved, and primers were released. The polyadenylated mRNA was reversetranscribed into cDNA. Full-length cDNA, along with cell barcode identifiers, was PCRamplified, and sequencing libraries were prepared and normalized.