A time-resolved, multi-symbol molecular recorder via sequential genome editing - Nature

, by increasing recording capacity and/or by modelling it during tree reconstruction. Although challenging to engineer, a generic recorder locus would allow us to take full advantage of DNA as an in vivo digital recording medium, for example, not only to characterize wild-type development, but also to enable systematic comparison of the developmental histories of wild-type and mutant individuals.

For lentivirus generation, approximately 300,000 HEK293T cells were seeded in each well of a six-well plate and cultured to 70–90% confluency. The lentiviral plasmid was transfected into cells along with the ViraPower lentiviral expression system , following the recommended protocol from the vendor. Lentivirus was collected following the same protocol, concentrated overnight using Peg-it Virus Precipitation Solution and used within 1–2 days to transduce HEK293T cells without a freeze–thaw cycle.

For transposase integration, 500 ng of cargo plasmid and 100 ng of Super piggyBac transposase expression vector were mixed and prepared with transfection reagent following the recommended protocol from the vendor and then transfected into confluent 24-well plates.

To append 10-bp unique molecular identifiers , we performed PCR in three steps: first, genomic DNA was linearly amplified in the presence of 0.04 to 0.4 µM of a single forward primer in two PCR cycles using KAPA Robust polymerase.

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