This SMT_VIDEOSreadme.txt file was generated on 2021-08-25 by Alex McCann GENERAL INFORMATION 1. Title of Dataset: Single Molecule Tracking Videos – SOX18 and its dominant-negative mutant SOX18RaOp 2. Author Information: A. Principal Investigator Contact Information Name: Mathias Francois Institution: Centenary Institute, University of Sydney Email: m.francois@centenary.org.au B. Alternate Contact Information Name: Alex McCann Institution: Institute for Molecular Bioscience, The University of Queensland Email: a.mccann@imb.uq.edu.au 3. Date of data collection: The data was collected between 2018-06-18 and 2020-02-20. 4. Geographic location of data collection: Data was collected at the Queensland Brain Institute (QBI) at the University of Queensland, QLD 4072 Australia DATA & FILE OVERVIEW 1. File List: VIDEO_S1_SOX18_VS_SOX18RaOp_20ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18RaOp in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2018-06-27, 2018-06-28 and 2018-07-10. VIDEO_S2_SOX18_VS_SOX18RaOp_500ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18RaOp in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2018-06-27, 2018-06-28 and 2018-07-10. VIDEO_S3_SOX18_WITH_SOX18RaOp_20ms .avi file showing the dynamics of HALO-SOX18 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 3:1 or 1:1 ratio in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2018-07-18, 2018-07-20 and 2018-07-24. VIDEO_S4_SOX18_WITH_SOX18RaOp_500ms .avi file showing the dynamics of HALO-SOX18 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 3:1 or 1:1 ratio in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2018-07-18, 2018-07-20 and 2018-07-24. VIDEO_S5_SOX7_20ms .avi file showing the dynamics of HALO-SOX7 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2020-2-12, 2020-02-14 and 2018-02-24. VIDEO_S6_SOX7_500ms .avi file showing the dynamics of HALO-SOX7 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2020-2-12, 2020-02-14 and 2018-02-24. VIDEO_S7_SOX17_20ms .avi file showing the dynamics of HALO-SOX17 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-08-16, 2019-08-22 and 2019-08-23. VIDEO_S8_SOX17_500ms .avi file showing the dynamics of HALO-SOX17 in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-08-16, 2019-08-22 and 2019-08-23. VIDEO_S9_SOX18DIM_20ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18DIM in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-07-19, 2019-07-25 and 2019-08-01. VIDEO_S10_SOX18DIM_500ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18DIM in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-07-19, 2019-07-25 and 2019-08-01. VIDEO_S11_SOX18AH1_20ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18AH1 in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-07-10, 2019-07-12 and 2019-07-17. VIDEO_S12_SOX18AH1_500ms .avi file showing the dynamics of HALO-SOX18 and HALO-SOX18AH1 in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-07-10, 2019-07-12 and 2019-07-17. VIDEO_S13_MEF2C_500ms .avi file showing the dynamics of HALO-MEF2C in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (500 ms acquisition for 500 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-06-26, 2019-07-03 and 2019-07-05. VIDEO_S14_MEF2C_20ms .avi file showing the dynamics of HALO-MEF2C in the presence of untagged-SOX18 or untagged-SOX18RaOp in a 1:1 ratio in the nucleus of HeLa cells (20 ms acquisition for 6000 frames). This .avi file was generated using a representative cell for each condition which is a part of a raw dataset that was collected on 2019-06-26, 2019-07-03 and 2019-07-05. METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: Immediately prior to imaging cells were washed twice and replaced with Fluorobrite DMEM (Glibco) imaging media. JF549 dye was a gift form Dr. Luke Lavis (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States). 2 drops/mL of NucBlue Live ReadyProbes Reagent (Hoechst 33342) was added directly to the media and cells were incubated for 5 min at 37 oC with 5 % CO2, prior to adding 2 nM of JF549 Halo-tag dye directly to the media and cells and incubation for a further 15 min at 37 oC with 5 % of CO2. Following incubation, cells were washed twice and replaced with Fluorobrite DMEM (Glibco). Images were acquired on an Elyra single molecule imaging (PALM/STORM) total internal reflection fluorescence (TIRF) microscope, with an Andor 897 EMCCD camera, SR Cube 05 RL – BP 420-480 / BP570-640 / LP 740 filter set and 100 X oil 1.46 NA TIRF objective using ZEISS ZEN blue software. Cells were imaged using a 561 nm excitation laser (power = 11.6 µW oblique illumination, 1.96 mW epi illumination; power density = 0.728 W/cm2 oblique illumination, 123.24 W/cm2 epi illumination) with a high-power TIRF filter (TIRF_HP). Using these parameters, we performed two different acquisition techniques; fast SMT which uses a 20 ms acquisition speed to acquire 6000 frames without intervals, and slow SMT which uses a 500 ms acquisition speed to acquire 500 frames without intervals. A low laser power was used to achieve a good signal-to-noise ratio with minimal photobleaching during imaging. Target and surrounding cells were prebleached for approximately 3 minutes (total across cells) prior to imaging to reduce the density of HALO-tagged molecules, background fluorescence, and the fluorescence interference from surrounding cells. 2. Methods for processing the data: Fast (20 ms acquisition, 6000 frames) and slow (500 ms acquisition, 500 frames) SMT raw image stacks were cropped in ImageJ to reduce their filesize and therefore minimize analysis time. Example image stacks for each condition were subsequently converted into AVI files using ImageJ in order to generate single molecule tracking videos.