GSM8515100: liver, CCl4+IPA; Mus musculus; RNA-Seq
Liver tissues were dissociated into single cells using a solution of 0.35% collagenase IV, 2 mg/ml papain, and 120 U/ml DNase I at 37°C with shaking for 20 minutes. Digestion was halted with 1× PBS/10% FBS, followed by filtration through a 70-30 µm strainer and centrifugation at 300g for 5 minutes at 4°C. The cell pellet was resuspended in 100 µl 1× PBS (0.04% BSA) and treated with 1× RBC lysis buffer (MACS 130-094-183), then centrifuged. Dead cells were removed using the Miltenyi® Dead Cell Removal Kit. The final cell suspension was washed, centrifuged, and resuspended in 50 µl 1× PBS (0.04% BSA). Viability (>85%) was confirmed with trypan blue exclusion, and cell concentration was adjusted to 700-1200 cells/µl using a hemocytometer or automated counter Following the manufacturer's instructions for the 10X Genomics Chromium Single-Cell 3' kit (V3), the dissociated cells were loaded into the 10X Chromium instrument for cell capture. Libraries were sequenced on an Illumina NovaSeq 6000 system (paired-end multiplexing run, 150 bp) at a minimum depth of 20,000 reads per cell (LC-Bio Technology Co., Ltd., Hangzhou, China).