Circadian Imaging of PER and CRY Proteins in U-2 OS cells

U-2 OS knock-in cells were genereated using CRISPR to express CRY1, CRY2, PER1 PER2 fused to either mScarlet-I or mClover3 from the endogenous promoter. All cells were transduced with H2B-iRFP using lentivirus to allow segmentation and tracking of nuclei.

Additionally, cells were transduced with lentivirus expressing shRNA against either FBXL3 (pGIPZ V2LHS_254986) or a nontargeted control shRNA.

===Raw Data===

==Dataset Dex==
Cells of selected clones were imaged for ~3days after synchronization with dexamethasone (1µM, 20min) at an imaging interval of 1 picture per hour
==Dataset CHX/Main==
In 3 experiments, cells were imaged for 68 hours with an imaging interval of 1 picture per hour.
After 44-48 hours, 20 µg/ml CHX was added to a subset of cells to inhibit protein production.
For assessment of fotobleaching, a subset of cells were imaged 20 times within 1 hour.

===Processed Data===
Cells were segmented and tracked using Cell Profiler. Position data, and background subtracted, mean nuclear fluorescence per nuclei and time point were extracted for RFP, YFP and iRFP channel.
A quality control step (Python Script) sorted out segmentation/traking errors, cells that leave the imaging area, and short time series.

===Data Analysis===
==Dataset Dex==
Circadian Parameters were analysed from tp 25 on
==Dataset CHX==
The first part of timeseries data was used to assess circadian parameters (e.g. rhythmicity, phase, period, amplitude), the second part to determine protein stability of the respective fluorescent fusion protein, which were correlated in subsequent analysis steps.