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Graphical abstract
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Figure 1. Schematic representation of the entire method pipeline
In Part 1 and 2 we describe how to prepare Xenopus laevis demembranated sperm nuclei and Low-Speed Supernatant Egg Extract (LSS), respectively. Part 3 details how to perform the large-scale chromatin isolation and FLAG immunoprecipitation of replisomes. Finally, Part 4 describes how to evaluate sample quality via silver staining and negative staining EM, as well as how to prepare grids for cryo-EM image acquisition (Schematics and EM density from
EMD-18195
).
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Figure 2. Schematic representation of the major steps of demembranated sperm nuclei preparation
Frogs are euthanized, testes dissected and cleaned. Subsequently, testes are finely chopped with a razor blade, filtered, aliquoted and stored appropriately.
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Figure 3. Schematic representation of the major steps of LSS preparation
After injection of Folligon and Chorulon, frogs will lay eggs. Bad quality eggs are discarded together with any debris (feces, shed skin). Eggs are extensively washed, dejellied and apoptotic eggs removed with a plastic Pasteur pipette. Clean eggs are aliquoted into tubes, packed and spin crushed. The central layer is collected using a syringe and clarified via ultracentrifugation. Finally, the top layer containing fats is discarded and the extract collected from the top, making sure that the bottom dark layer is not perturbed at any time.
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Figure 4. Handling and injection of Xenopus laevis
Both females and males are injected into the dorsal lymph sac.
(A) The area for injection corresponding to the dorsal lymph sac is highlighted in green. Red indicates the lateral line system, resembling stitches on the frog skin. These are sensorial organs, therefore, avoid injecting directly around them.
(B) Pictures of how to handle a frog for injection and how to expose the area to inject.
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Figure 5. Images of demembranated sperm nuclei preparation
(A) Xenopus laevis male with highlighted the area where testes are located. Insert: Picture of testes surrounded by other tissues.
(B) Dissected testis with blood vessels (i). Cleaned testis (ii), ready to be chopped (iii) and filtered (iv). The filtered material (v) is subsequently centrifuged (vi) and eventual erythrocytes (vii) removed before collection and storage. Scale bar 1 cm.
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Figure 6. Images of eggs collection
(A) Good quality eggs are inactivated and arrested in meiosis II metaphase. They are individual, with clear dark animal pole and light vegetal pole, and surrounded by clear buffer.
(B) Bad quality eggs may be characterized by discolored or speckled yolks, white bloated eggs, stringy eggs, small size, dirty jelly coats with debris adhering to the surface.
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Figure 7. Images of the major steps of LSS preparation
(A) Rinsed eggs with jelly layer still present.
(B) Eggs after dejellying are compacted at the bottom of the beaker.
(C) Dejellied eggs transferred into a tube.
(D) Compacted eggs with presence of buffer and apoptotic eggs on the top of the tube.
(E) Floating apoptotic eggs (white) are removed using a plastic Pasteur pipette.
(F) Compacted eggs ready to be spin-crushed.
(G) Egg extract, after crushing spin. (i) lipid layer, (ii) crude cytoplasm, (iii) yolk platelets.
(H) Egg extract, after clarifying spin. (i) lipid layer, (ii) membranous layer, (iii) cytoplasm, (iv) mitochondria, residual yolk platelets and insoluble material.
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Figure 8. Negative staining of the grids
(A) Set-up for negative staining with grids in tweezers and UA droplets on parafilm.
(B) Staining of a grid on the first UA droplet.
(C) Blotting away residual UA solution.
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Figure 9. Set-up for plunge freezing
(A) Humidifier attached to the Vitrobot.
(B) Circular blotting paper attached to blotting pads inside the Vitrobot chamber.
(C) Assembled vitrification container set.
(D) A grid plunged into liquid ethane on the Vitrobot.
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Figure 10. Quality evaluation of demembranated sperm nuclei
(A) Sperm count with hemocytometer in bright field with 20x objective. Scale bar 50 μm, inset 25 μm.
(B–E) Fluorescence images of nuclear formation and development. The reaction started when sperm nuclei are added to egg extract released in interphase (0 min), and was followed over 60 min at indicated times: 15 min (B), 30 min (C), 45 min (D) and 60 min (E). Sample stained with Hoechst 33258 (5 μM final concentration), scale bar 20 μm.
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Figure 11. Quality evaluation of Xenopus egg extract
(A) DNA synthesis was assayed via α32P-dATP incorporation detection.
(B) Time course of DNA replication reaction with chromatin associated factors. The reaction was assembled with and without CaCl2. Chromatin was isolated at specific time points during DNA replication, separated via SDS–PAGE and immunoblotted with antibodies against main replication factors, while the lower portion of gel was stained with Coomassie blue to identify histones.
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Figure 12. Assessment of purified replisomes after IP
(A) Silver-stained gel of the large-scale FLAG-IP samples with CMG components highlighted. Adapted from.
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(B) CMG and other co-immunoprecipitated proteins from the IP sample, detected by mass spectrometry. Selected replication factors are presented with molecular weight and total spectral count. Full data available on PRIDE. (∗Xenopus tropicalis proteins). Adapted from.
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Figure 13. Multiple sample applications on a grid
(A) Negative stain EM micrograph after single sample application on the grid.
(B) Negative stain EM micrograph after three consecutive sample applications on the grid.
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