MNase assay with EZ Nucleosomal DNA prep Kit

Nucleosomal DNA preparation

(EZ Nucleosomal DNA prep Kit with MNase (D5220 Zymo Research)

Nuclei Isolation Procedure

Note: All steps are carried out on ice.

1. Lyse freeze tissue (e.g. 20 guts) in 80 μl Nuclei Prep buffer (Zymo research), then add 120 μl Nuclei Prep buffer.
2. Incubate on ice for 5 min.
3. Pellet nuclei at 500 g for 2 min.
4. Wash the pellet in 100 μl Digestion buffer.
5. Pellet nuclei at 500 g for 2min.
6. Repeat washes one more time.

Enzymatic Treatment

1. Gently resuspend nuclei pellet in 120 μl Digestion buffer. And take 5 μl for DNA measurement (Qubit dsDNA HS Kit). 
2. Take the same amount of DNA for all samples, then add Digestion buffer to 110 μl. 
3. Then take 20 μl and add to 4 μl of 5X stop Buffer (Input).
4. Take 1 μl MNase (1U/μl) and add to 39 μl Digestion buffer, then add 1 μl (0.0025 U) MNase dilution per sample and mix by pipetting a couple of times. (! The MNase final concentration for digestion needs to be optimised with the trial sample.)
5. Stop reaction at 1, 2, 5, and 10 mins by taking 20 μl of reaction and adding it to 4 μl of 5X stop Buffer. Vortex sample to mix 5 sec.

Nucleosomal DNA Purification

Note: ensure ETOH has been added to DNA Wash Buffer.

1. Add a 5:1 ratio of DNA Binding buffer to the stopped reaction. E.g. 120 μl to a 24 μl reaction.
2. Mix well by vortexing.
3. Load the sample into a Zymo-Spin IIC column in a Collection Tube.
4. Spin for 30 sec at 12000 g
5. Discard flow through from the Collection Tube.
6. Add 300 μl DNA Wash Buffer (Qiagen) and spin for 30 sec 12000 g, and Discard flow through.
7. Repeat step 6 once.
8. Spin the empty column for 1 min at 12000 g to remove any residual buffer.
9. Transfer the spin column to the fresh tube. 
10. Add 20 μl DNA elution buffer (Qiagen) and let stand for 2min.
11. Spin for 1 min at 12000xg to elute nucleosomal DNA.

Run samples on TapeStation using High Sensitivity D5000 ScreenTape