Preparation

1. Clean the bench top and dissection area with 70% ethanol, followed by RNaseZAP to remove RNases.

2. Tissue Collection

     a. Rapidly remove rat brains and flash-freeze in pre-chilled 2-methylbutane (dry ice or liquid nitrogen) for 60 seconds, then store at -80°C until sectioning.

     b. Embed brains in optimal cutting temperature medium (OCT) and section with a cryostat (250 μm sections).

     c. Micropunch sections containing the auditory cortex (AC) with a 1 mm diameter micropuncher.

     d. Transfer to ice-cold centrifuge tubes and store at -80°C until nuclei isolation.

 

Procedure

Note:  Keep all tools, materials, and reagents on ice. Pre-cool the swinging bucket centrifuge to 4°C

1. Combine frozen micropunches and briefly thaw on wet ice.

2. Transfer to a 1.5 mL tube with 400 μl chilled lysis buffer.

3. Dissociate the tissue on ice using a plastic pestle homogenizer to grind the tissue manually until homogeneous.

4. Incubate on ice for 10 minutes.

5. Pass micropunches through pre-chilled Nuclei Isolation Column* (spin column) by centrifuging at 500 x g for 3 minutes at 4°C in a swinging bucket centrifuge to pellet nuclei.

6. Remove the supernatant without disrupting the pellet.

7. Wash nuclei-pellets with 1 mL chilled Nuclei Wash and Resuspension Buffer by gently pipette mixing 10 times.

8. Filter nuclei through a 30 μm strainer into a 1.5 mL centrifuge tube (Protein LoBind tube highly recommended)

9. Centrifuge at 500 x g for 3 minutes at 4°C in a swinging bucket centrifuge to pellet nuclei.

10. Remove the supernatant without disrupting the pellet.

11. Resuspend nuclei in 500 μL Nuclei Wash and Resuspension Buffer by gently pipetting 10 times.

12. Filter twice through a 30 μm strainer, collecting the filtrate in a new tube each time.

13. Count nuclei using a preferred accurate method.

Note: In our experience, nuclei are much stickier than whole cells. For researchers making the single-nuclei suspension for the first time, we suggest confirming that the sample contains mostly single nuclei. To do this, take 10 μL of the sample and stain it with Hoechst, DAPI, or AOPI for more than 5 min, place it on a hemocytometer, and visualize it under a 20X and 40X objective on an epifluorescence microscope. A best practices resource for hemocytometer use, see: https://www.hemocytometer.org/

14. Aliquot nuclei and centrifuge at 200 x g for 10 min at 4 °C. 

15. Proceed to the appropriate Parse Biosciences Nuclei Fixation Kit User Guide (either standard Nuclei Fixation, Low-Input Nuclei Fixation). 

 

Notice

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