GSM9029989: 2404_2024-GEX, Transplanted tomato positive larry-barcoded HSCs, bio reps 1-4; Mus musculus; RNA-Seq
In both isolations (donor and recipient): bone marrow was collected from the femur, tibia, pelvis, sternum and column through mechanical bone crushing. Cells were filtered through a 100vm 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), PE anti-mouse CD201 (EPCR) (Biolegend, Catalog no. 141504), PE/Cy5 anti-mouse CD150 (Biolegend, Catalog no. 115912) and APC/Cy7 anti-mouse CD48 (Biolegend, Catalog no. 103432). Cells were then washed and HSCs were purified through Flow Cytometry Cell Sorting using a CytoFLEX SRT (Beckman Coulter, Catalog no. C71883). From each recipient mice, donor Lin-, LSK and Mk TdTomato+ and TdTomato- were extracted and labelled  with a specific Biolegend TotalSeq B antibody (one per mouse) to do Feature Barcoding in order to multiplex the mice (independently of the labeling). 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 4 different mice) were loaded each of them in a run for performing single-cell libraries using Chromium Single Cell 3' Reagent Kits (v3) following the manufacturer's guidelines (10X) Genomics). Remnants of amplified cDNA were used to perform two sequential PCRs in a nested-PCR design to enrich for the LARRY-barcode transcripts and ensure correct coverage of the full barcode sequence.