title: GSM8525130: LSKs isolated from mice fed LFD; Mus musculus; RNA-Seq
library_construction_protocol: Total BM cells were isolated from mice by crushing the femur, tibiae, and pelvic bones in staining medium composed of Hank's buffered saline solution (HBSS) containing 3% heat-inactivated fetal bovine serum (FBS). Single-cell suspensions were purified on a Ficoll gradient, and then red blood cells were. Total BM cell counts were obtained using an automated cell counter. BM cells were lineage depleted using lineage microbeads and a MACS separator. Lineage-depleted BM cells were then blocked using purified rat IgG diluted 1:100 in staining buffer. Cells were then incubated with the following lineage antibodies conjugated to PE-Cy5: Gr1 (1:800), CD11b (1:800), CD3 (1:400), TER119 (1:400), CD5 (1:800), B220 (1:800), CD4 (1:800), CD19 (1:800), CD8a (1:800), in addition to the following antibodies staining for HSPC markers: c-Kit-APC-Cy7 (1:800) and Sca1-BV421 (1:400). LSK cells were sorted into HBSS + 3% FBS, allowed to rest on ice for 30 minutes, then were sequenced. Library was performed according to the Single Cell 3' v3 protocol (10x Genomics).