Freedom evo liquid handler

Cells were moved from agar plates into liquid 384‐well plates using the RoToR bench‐top colony arrayer (Singer Instruments). Liquid cultures were grown overnight in synthetic medium with 2% glucose (SD) in a shaking incubator (LiCONiC Instruments) at 30°C. A Tecan freedom EVO liquid handler (Tecan), which is connected to the incubator, was used to back‐dilute the strains to ∼ 0.25 OD₆₀₀ in plates containing the same medium. Plates were then transferred back to the incubator and were allowed to grow for 4 h at 30°C to reach logarithmic growth phase. The liquid handler was then used to transfer strains into glass‐bottom 384‐well microscope plates (Brooks Bioscience) coated with Concanavalin A (Sigma‐Aldrich) to allow cell adhesion. Wells were washed twice in a low fluorescence synthetic medium (Formedium) to remove floating cells and reach a cell monolayer. Plates were then transferred into the automated microscopy system using a KiNEDx robotic arm (Peak Robotics).
Imaging was performed using an automated Olympus SpinSR system using a Hamamatsu flash Orca 4.0 camera and a CSUW1‐T2SSR SD Yokogawa spinning disk unit with a 50 μm pinhole disk. Images were acquired using a 60× air lens NA 0.9 (Olympus), 100 mW 488 nm OBIS LX laser system (Coherent), GFP Filter set [EX470/40, EM525/50] (Chroma).
Images were manually inspected using Fiji‐ImageJ software (Schindelin et al, 2012 (link)).

SNP Chromosome microarray analysis was implemented to confirm genome integrity by examining genomic DNA extracted from hiPSC cells. The Illumina Human CytoSNP-12 assay (Illumina, USA) was performed according to manufacturer's protocols. In summary, 300 ng of hiPSC gDNA underwent whole-genome amplification overnight at 37°C, before fragmentation, purification, and loading onto a Human CytoSNP-12 BeadChip for overnight hybridisation at 48°C. The next day, the BeadChip underwent single base-pair extension and staining using the illumina Automation Control program (Illumina, USA) on the Tecan Freedom EVO liquid handler (Tecan, Switzerland). SNP microarray analysis was performed using BlueFuse Multi v4 (Illumina, USA), with the variant calling algorithm settings as follows: dosage log ratio quality metric (DLRDev) < 0.24, ≥8 consecutive adjacent SNP probes consistent with the copy number change detected, with LogR ratios being > +0.2 and < -0.3 for duplication and deletions, respectively.

All robotic workflows were carried out on a Freedom Evo Liquid Handler (Tecan, Switzerland) with integrated centrifuge (Hettich, Germany) and plate reader (Tecan, Switzerland). Experiments, which combined microcultivation and robotic workflows, were conducted using an in-house device control system. This system was used to continuously monitor BioLector data using the Python package bletl [51 (link)] and carry out sampling and sample processing workflows based on previously defined triggers (e.g., cultivation time). The necessary pipetting worklists were written with Python package robotools [52 (link)]. During automated sampling procedures, 800 µL sample were taken from the given cultivation well, added to a DWP that contained a defined amount (up to 75 µL) of 4 M sulphuric acid and incubated for 30 min, if necessary. After incubation, the samples were centrifuged at 4000×g at 4 °C for 6 min. 500 µL supernatant was transferred to a DWP containing a defined amount (up to 50 µL) of 8 M NaOH, if necessary. The supernatants were placed on a cooled tray on the robotic deck and analyzed for antimicrobial activity.