GSM9039058: TPPP3 GEM-X FB; Mus musculus; RNA-Seq
Bone marrow was collected from the femur, tibia, pelvis, sternum and column through mechanical bone crushing. Cells were filtered through a 100um strainer and resuspended in PBS with 2% fetal bovine serum (FBS), followed by lysis of red blood cells using RBC lysis buffer (Biolegend, Catalog no. 420302). Cells were then enriched for c-Kit expression using CD117 MicroBeads (Miltenyi Biotec, Catalog no. 130-091-224). The resulting cKit-enriched fraction was washed and stained with fluorescently labeled antibodies: APC anti-mouse CD117, clone ACK2 (Biolegend, Catalog no. 105812), PE/Cy7 anti-mouse Ly6a (Sca-1) (Biolegend, Catalog no. 108114), Pacific Blue anti-mouse Lineage Cocktail (Biolegend, Catalog no. 133310). Cells were then washed and HSCs were purified through Flow Cytometry Cell Sorting using a CytoFLEX SRT (Beckman Coulter, Catalog no. C71883). HSCs were labelled  with a specific Biolegend TotalSeq B antibody (one per mouse per condition) to do Feature Barcoding in order to multiplex the Tppp3 labelled and non-labelled mice. TdTomato positive stained cells were sorted on the same tube and TdTomato negative stained cells were sorted on other tube. These two tubes (with sorted TdTomato positive or negative from the 2 different mice) were loaded in a run for performing single-cell libraries using Chromium Single Cell 3' Reagent Kits (v3) or GEM-X following the manufacturer's guidelines (10X Genomics).