DOI: http://dx.doi.org/10.7554/eLife.04766.003 To test whether S phase is optimal for HDR in HEK293T cells, six reversible chemical inhibitors were used in parallel experiments to synchronize HEK293T cells at G1, S and M phases of the cell cycle, followed by release prior to nucleofection with Cas9 RNP.
Fig. 3 Schematic representation of the relevant distances in the experiment (a) together with a schematic representation of the trigger scheme (b) needed to synchronize the experiment.
To synchronize each experiment, the baseline was set automatically by the software.
Before the beginning of the experiment, time informations are broadcasted (UDP) to synchronize the internal clock of each device; time error is less than ten milliseconds.
To synchronize the whole experiment, at the F1 generation, brother sister mating, as well as, crosses between a single O57 male and a single W29 female, and a single O57 female and a single W29 male were performed.
Unlike experiments 1 and 2 where we sought to synchronize neural activity with in-phase stimulation and enhance working memory, experiment 3 tested whether a desynchronizing or anti-phase montage could impair working memory.
In order to synchronize cells before experiments, all cultures were serum-starved overnight in antibiotic free and serum free DMEM.
Because the breathing cues that we used for the fMRI experiments were visual, and therefore might have distracted from the visual task, for the behavioral experiment we instead used auditory breathing cues to synchronize inhalation with the olfactometer.
We observed that the use of SSH protocols and Fast Ethernet connectivity allows nodes to synchronize the start of an experiment with high accuracy, being all instructions read at once; afterwards, the testbed relies on individual clocks to synchronize instructions throughout the remaining time of an experiment.
To synchronize cultures for growth curve experiments, trophozoites were selected by gelatin flotation method [30] 24 h before setting up experiments with ring stage parasites.
