LSK immunophenotype hematopoietic stem cells after irradiation at day points in 3 weeks

In this study, we aimed to investigate the differentiation dynamics of LSK (Lin-Sca1+c-Kit+) cells following radiation injury. LSK cells, which are a critical component of the hematopoietic stem and progenitor cell population, play a vital role in the regeneration and repair of the hematopoietic system after damage. By utilizing these cells, we sought to understand the mechanisms underlying their response to radiation, focusing on their capacity for self-renewal and differentiation into various blood cell lineages. To perform 10X Genomics single-cell sequencing on mouse bone marrow LSK cells, begin by euthanizing the mouse following ethical guidelines and extracting the femurs and tibias. Flush the bone marrow using a syringe with a suitable buffer, such as PBS with 2% FBS, to obtain a single-cell suspension. Filter this suspension through a 40 µm cell strainer to remove debris and clumps. Next, enrich the LSK (Lineage^-, Sca-1^+, c-Kit^+) cells using magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS). Count the cells and assess viability using trypan blue staining or a similar method. Adjust the concentration of LSK cells to the required density, typically 700-1200 cells/µL, for optimal performance in the 10X Genomics Chromium Controller. Load the single-cell suspension, reagents, and gel beads into the appropriate wells of a 10X Genomics Single Cell 3' Chip and run the chip in the Chromium Controller to encapsulate single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs). Perform reverse transcription within the GEMs to generate barcoded cDNA from the mRNA of individual cells, then break the emulsion and clean up the barcoded cDNA. Amplify the cDNA via PCR and assess the quality and quantity using an Agilent Bioanalyzer or similar instrument. Construct sequencing libraries from the amplified cDNA, following the 10X Genomics protocol. Sequence the libraries on an Illumina platform, such as NovaSeq or NextSeq, according to the recommended read length and depth for single-cell RNA-seq. Finally, use the 10X Genomics Cell Ranger pipeline to process the raw sequencing data, perform alignment, and generate a gene expression matrix. Analyze the data using bioinformatics tools to identify cell populations, gene expression patterns, and potential changes in cell fate or differentiation pathways.