Genomic DNA was extracted from sorted hematopoietic cells and amplified using Phusion PCR Master Mix (Thermo Scientific, Waltham, MA). The polymerase chain reaction (PCR) was stopped at the exponential phase, and the PCR product was purified using AMPure XP beads (Beckman Coulter A63880) before high-throughput sequencing. The sequencing data were analyzed as previously described29 (link),33 (link),34 (link). Barcode data were analyzed using our customized Python scripts32 –34 (link). We filtered out clones with more than 0.5% white blood cell (WBC) abundance in one cell type at one-time point and absent in all other cell types and time points. Sequencing data were combined with FACS data to calculate the clonal abundance as follows:
Clonal abundance = 100% × (each cell population (Gr, B, CD4 T, or CD8 T cells)% WBCs) × (donor% each cell population) × (GFP% donor cells) × (number of reads for each barcode)/(total reads of all barcodes).
Clonal abundance = 100% × (each cell population (Gr, B, CD4 T, or CD8 T cells)% WBCs) × (donor% each cell population) × (GFP% donor cells) × (number of reads for each barcode)/(total reads of all barcodes).
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