Fig. 1

In vitro and in vivo validation of RNA barcoding approach for stable, long-term clonal kinetics studies of hematopoiesis. A Experimental setup for in vitro validation of quantitative RNA-based clonal studies. The K562 cell line was used for the transduction, and single eGFP + cells were FACS-sorted and expanded to generate monoclonal calibration samples. Four clones were used to create samples, where cells carrying different barcodes were mixed in known ratios-spiked in clone contributing 0.1%, 1%, 5%, 10%, 20%, or 50% of the total mix. B Generalized linear model (GLM) correlation between the read count for barcoded transcript retrieved in calibration samples from cDNA or gDNA. C Detection of unequally mixed barcodes in cDNA and gDNA samples, ten barcoded clones of the BaF3 cell line were mixed in 1:10:10:50:100:100:200:200:200:250 ratios, the smallest clone contributing at 0.089%. Presented are results from 2 technical replicates. D Experimental setup to test the silencing of eGFP in VavCre x Rosa26tdTomato LSK cells. E–I eGFP + chimerism changes in tdTomato + anucleate cells and CD45.2 + (donor-derived) nucleated cells. To calculate the silencing in specific lineage, we subtracted eGFP + cells from CD45.2 + cells (nucleated cells-CD19 + , Mac1 + or CD4/8 + cells) or from tdTomato + cells (all donor-derived anucleate cells); shown are the results from a single experiment in 3 recipient mice