RNA-Seq of macrophages isolated from peripheral blood of Patient_003
The peripheral blood samples were first mixed with an equal volume of red blood cell lysis buffer. Pure PBMCs (peripheral blood mononuclear cells) suspension was obtained by centrifugation to remove the lysed red blood cells and their debris.The resulting single-cell suspension was collected and resuspended in a staining buffer containing fluorescent dye-labeled anti-CD11b antibodies and F4/80. The cells were then stained and fixed. After washing with PBS, macrophage populations were identified and sorted using a flow cytometer for subsequent culture and maintenance.Total RNA was extracted using TRIzol reagent, the DNA sample is randomly fragmented into small fragments with lengths ranging from 300 to 800 bp through enzymatic digestion. The sticky ends of the fragments are then blunted, and specific sequencing adapters are ligated to both ends of the DNA fragments. Subsequently, PCR amplification is performed to enrich the target DNA fragments with adapters, ensuring that the library fragment size meets the expected range. Quantitative PCR (qPCR) is used to accurately quantify the effective concentration of the library.Libraries were sequenced on a NovaSeq 6000 (150 bp paired-end). Samples are peripheral blood from tougue cancer patients.