title: LC4
organism: Homo sapiens
description: Protocols: Peripheral blood samples were collected from hepatocellular carcinoma (HCC) patients before and after curative therapy. Blood was drawn into EDTA tubes and processed within 2 hours to isolate peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque density gradient centrifugation. Not applicable. No tissue dissection was performed. PBMCs were isolated directly from peripheral blood. Not applicable. PBMCs were isolated directly from peripheral blood and were not cultured prior to library preparation. Peripheral blood samples were collected from patients both before and approximately one to three months after curative therapy (radiofrequency ablation or surgical resection). No in vitro treatment was applied to the PBMCs before sequencing. The raw sequencing data were demultiplexed and converted into FASTQ files using the cellranger mkfastq pipeline. The FASTQ files were subsequently processed with Cell Ranger to generate gene-barcode count matrices. Total RNA was not extracted separately. Single-cell suspensions of viable PBMCs were directly used for cDNA synthesis within the 10x Genomics Chromium single-cell 3′ gene expression protocol, which includes cell lysis and RNA capture inside oil droplets. Single-cell 3′ gene expression libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Reagent Kits v3 according to the manufacturer's instructions. Each library includes reverse transcription, cDNA amplification, fragmentation, end-repair, A-tailing, adapter ligation, and sample indexing.