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. Author manuscript; available in PMC: 2021 Jun 11.
Published in final edited form as: Cell. 2020 May 14;181(6):1410–1422.e27. doi: 10.1016/j.cell.2020.04.048

Figure 2: Multiple pulses of doxycycline can consecutively label lineages and enable phylogenetic tree reconstruction in embryonic stem cells.

Figure 2:

A. Chord plots of CARLIN alleles in the absence of doxycycline (Dox) and after one, two or three 6h pulses of Dox (R0–3, respectively). Color scheme as in Figure 1F.

B. Following one 6h round of Dox induction, cells were seeded at single-cell density and 8 colonies were picked for further outgrowth and Sanger sequencing. Following a second round of Dox, DNA from cells was collected and sequenced by Next Generation Sequencing (NGS). Schematic created with BioRender.

C. Mutations called in each of the 8 colonies from the CARLIN pipeline applied to the Sanger sequences. Colonies are colored according to the schematic in (B). Colonies 5, 7 and 8 share a common mutation.

D. (Left panel) The consensus tree, accounting for 95% of cells, obtained from 10,000 lineage reconstruction simulations applied to alleles pooled from all libraries (Methods; Supplementary Figure 4C). The color of a node and its branch to a parent corresponds to the NGS library in which the allele was observed. Leaves that connect to internal nodes of a different color correspond to false positives. (Centre panel) Sequence of each CARLIN allele visualized as in Figure 1C. (Right panel) Histogram of the number of cells in which each allele was detected. Colored bars correspond to NGS sequences which match a Sanger sequence.