Extended Data Fig. 9: Multiplex recording of CRE activity in embryonic stem cells and differentiating gastruloids.

(a) Log-scaled insertion proportions for 5-mer barcodes linked to the 98 synthetic CRE-driven ENGRAM recorders were highly reproducible across integration replicates for cultured mESC, as read out by amplification and sequencing of synthetic DNA Tape. (b) Log-scaled proportions for 5-mer barcodes linked to the 98 synthetic CRE-driven ENGRAM recorders are well correlated between the original plasmid pool and genomic integrations in the polyclonal mESC line. (c-e) Log-scaled barcode proportion ratios, as calculated from one pair of replicates vs. as calculated from another pair of replicates, for mESC vs. HEK293T cells (c), mESC vs. K562 cells (d), or K562 vs. HEK293T cells (e). Note that we corrected the mESC data for differences in relative abundances of recorders in the polyclonal mESC line vs. the plasmid pool used to transiently transfect K562 and HEK293T cells (as shown in Extended Data Fig. 9b), prior to performing these comparisons. As the same number of cells were sampled for recording, the lower reproducibility for comparisons involving K562 cells is likely secondary to lower transfection/editing efficiency, as shown in Extended Data Fig. 5a. (f) Stacked bar plot showing the proportion of 5-mer barcodes associated with each of the 98 synthetic CRE-driven ENGRAM recorders in cell lines and gastruloids. Recorder activities are presented in the order of their maximal proportion across all samples. Error bars correspond to standard deviations across 3 transfection replicates (in K562 and HEK293T cells), or 3 integration replicates in mESCs and 2 integration replicates for gastruloid time-windows. Note that these labels are representative of TF(s) thought to bind each motif, and it remains uncertain which TF(s) are driving the activity of each synthetic CRE recorder. See Supplementary Table 3 for the corresponding consensus motifs, and the full list of TFs associated with each motif by the JASPAR database.