Day 1: Nuclei isolation & chromatin denaturation

⏱️ 2 hours

<aside> 🌡️ Set the centrifuge and microfuge to 4°C. Set the water bath / heat block to 37°C, and another one to 62°C.

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• [ ] Pre-cool a 50 ml centrifuge to 4°C.
• [ ] Resuspend 1.5–2 g of cryo-ground plant material in 20 ml of chilled nuclei isolation buffer in a 50 ml centrifuge tube.
• [ ] Ensure the centrifuge tube lid is fastened and place the sample horizontally on ice and mix on a gyratory rocker at 50 rpm for 15 minutes.
• [ ] Pass the suspension through a 40 μm cell strainer into a fresh chilled 50 ml tube on ice.
• [ ] Once the sample has passed through the filter, rinse the original sample tube with a further 5 ml of chilled nuclei isolation buffer and filter through the same 40 μm cell strainer.
• [ ] Centrifuge for 20 minutes at 4°C using a centrifugation force appropriate for the genome size.
  • What is the appropriate centrifugation force for my genome?
• [ ] Carefully decant and discard the supernatant. Using a wide-bore pipette tip, gently resuspend the pellet in 1 ml of chilled nuclei isolation buffer.
• [ ] Add a further 19 ml chilled nuclei isolation buffer to the suspension and mix by swirling.
• [ ] Centrifuge for a further 10 minutes at 4°C using a centrifugation force appropriate for the genome size.
• [ ] Remove the supernatant, resuspend the pellet, and centrifuge again as the previous steps.
• [ ] Pre-cool a 2 ml centrifuge to 4°C.
• [ ] Carefully decant and discard the supernatant. Using a wide-bore pipette tip, gently resuspend the pellet in 1 ml of cold washing buffer. Transfer the sample to a fresh 2 ml centrifuge tube on ice. Rinse the original tube with a further 1 ml of cold washing buffer and transfer to the same 2 ml centrifuge tube.
• [ ] Centrifuge for 5 minutes at 4°C using a centrifugation force appropriate for the genome size.
• [ ] Aspirate and discard as much of the supernatant as possible without disturbing the pellet. Using a wide-bore pipette tip, gently resuspend the pellet in 2 ml of chilled 1X PBS.
• [ ] Centrifuge for 5 minutes at 4°C at 3000 g.
• [ ] Aspirate and discard the supernatant.