Figure 1.

Experimental design. Oligonucleotides consisting of a 6bp library ID followed by a 27-35bp high diversity random sequence barcode were inserted into a lentiviral vector flanked by polymerase chain reaction (PCR) primer sites. RM CD34⁺ hematopoietic stem and progenitor cell (HSPC) were mobilized into the peripheral blood (PB), collected by apheresis, enriched via immunoselection, transduced with the barcoded lentiviral library, and infused back into the total body irradiation (TBI) irradiated autologous macaque. After engraftment, PB and bone marrow (BM) samples were obtained, and various hematopoietic lineages were purified for barcode retrieval and analyses. Lineage cells and nucleated red blood cell (NRBC) were purified from the BM and/or PB. Colony-forming unit (CFU) derived from CD34⁺ BM cells cultured in semi-solid media, and mature red blood cell (RBC) and reticulocytes were enriched via depleting nucleated cells from the PB. DNA and/or RNA were extracted for barcode PCR, high-throughput sequencing, and custom data analysis.