Nuclei Isolation

Nuclei after PI staining

  1. Add NIB medium (CelLytic™ PN Isolation/Extraction buffer) to a new, clean, sterile, and pre-refrigerated 60 mm petri-dish.
  2. Collect fresh roots from seven day-old Arabidopsis plants by separating the root from the aerial part of the plant with a razor blade.
  3. Transfer the roots with forceps into the 60 mm Petri dish. Be sure to expose the roots to the NIB medium. Note: Remove any piece of aerial plant tissue to avoid any contamination of the root samples.
  4. Finely chop the roots with a razor blade for about 5 minutes.
  5. Add additional NIB medium if needed to keep the root tissues moist .
  6. Incubate 15 minutes in the cold room using a gentle horizontal shaking (i.e., platform rockers).
  7. In an ice bucket, place a 50 ml Falcon tube with a 30 µm strainer mounted on a 40 µm strainer at the top.
  8. Pre-wet the 40 and 30 µm strainers with 500 µl of NIB medium before to filter the nuclei suspension. The tube should be slightly tilted on its side.
  9. Using wide-bore tips, slowly pipet the chopped roots-NIB mixture from step 6 onto the pre-wet strainers.
  10. Using wide-bore tips, slowly rinse the plate with 500 µl NIB medium to collect the remaining nuclei.
  11. Add the extra 500 µl chopped root-NIB medium onto the strainers to collect the remaining nuclei and to remove debris.
  12. The filtered nuclei sample is ready for fluorescence-activated nuclear sorting.
  13. After sorting, centrifuge nuclei at 1000 × g for 10 minutes at 4°C.
  14. Remove the supernatant.
  15. Add 3ml of PBS+.
  16. Gently resuspend the nuclei.

Reference

Thibivilliers S,  Anderson D,  Libault M (2020). Isolation of Plant Root Nuclei for Single Cell RNA Sequencing. Current Protocols in Plant Biology. 5(4). doi.org/10.1002/cppb.20120.