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Supplemental Material for Cahoon and Libuda, 2021

dataset
posted on 30.08.2021, 15:40 by Cori K. Cahoon, Diana E. Libuda

Figure S1. Diagram of live imaging mounting and post-imaging recovery. For a comprehensive description see the Methods section “Conditional immobilization for live imaging”.


Table S1. Primer sequences used for CRISPR/Cas9 tagging unc-104, unc-18, and unc-52 with auxin inducible degron tag


Movie S1. Brightfield 10x timelapse movie of an immobilized hermaphrodite worm. A hermaphrodite worm containing unc-18::AID*; rgef-1p::TIR1 that was immobilized on 1mM auxin and mounted under an agar pad. Images were captured every 90 seconds for 60 minutes.

Movie S2. Autofluorescence in an immobilized L1 worm. A L1 worm containing unc-18::AID*; rgef-1p::TIR1 that was immobilized on 1mM auxin and mounted under an agar pad. Timelapse images were taken at 60x magnification and images were captured every 2 minutes for 60 minutes.

Movie S3. mCherry::H2B in an immobilized L4 hermaphrodite worm. Timelapse images were taken at 60x magnification and images were captured every 2 minutes for 44 minutes.

Movie S4. SYP-2::GFP in an immobilized hermaphrodite worm. Timelapse images were taken at 60x magnification and images were captured every 5 minutes for 65 minutes. Movie was stabilized for viewing (see Methods for details).

Movie S5. Whole germline view of SYP-2::GFP in an immobilized hermaphrodite worm. Timelapse images were taken at 60x magnification and images were captured every 5 minutes for 2 hours. The start of the germline occurs out of view of the camera on the left and germline progresses from left (transition zone/early pachytene) to right (late pachytene/diplotene) within the movie. Then, the germline makes a U-turn bend, which is out of view of the camera. The germline comes back into view indicated by the arrowhead showing the appearance of the diakinesis oocytes on the bottom right of the movie, which are moving from right to left. Movie was stabilized for viewing (see Methods for details).

Movie S6. Brightfield 20x timelapse movie of an immobilized male worm. A male worm containing unc-18::AID*; rgef-1p::TIR1 that was immobilized on 10mM auxin and mounted under an agar pad. Images were captured every 90 seconds for 60 minutes.

Movie S7. SYP-2::GFP in an immobilized male worm. Timelapse images were taken at 60x magnification and images were captured every 5 minutes for 65 minutes. Movie was stabilized and corrected for photobleaching for viewing (see Methods for details).

Movie S8. Whole germline view of SYP-2::GFP in an immobilized male worm. Timelapse images were taken at 60x magnification and images were captured every 5 minutes for 2 hours. Arrowhead indicates the approximate start of the germline within the mid-bottom region of the movie. Germline progression initially starts off moving to the left, then the germline makes a U-turn bend at the transition zone on the left side of the movie. After this bend, the germline progression moves from the left (early pachytene) to right (late pachytene/diplotene) within the movie. The end of the germline (condensation and mature sperm) occurs farther off to the right outside of the camera view at this magnification. Movie was stabilized and corrected for photobleaching for viewing (see Methods for details).


History

Article title

Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion