title: GSM8518011: WT, scRNA; Mus musculus; RNA-Seq
library_name: GSM8518011
library_construction_protocol: 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 with  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 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  Illumina 3000 platform.
identifiers.namespace: GEO
identifiers.id: GSM8518011_r1