DNA Fragmentation in ChIP exo

The aim of this part of the ChIP-exo protocol is to break open the cells so the content can be exposed and the DNA can be sheared into small fragments. It consists of two different parts:

• Cell lysis: the bacterial cell walls will be broken. Three different solutions should be added:

  • Lysis buffer: it will create an adequate environment for the lysozyme and keep the pH stable.

  • Protease inhibitor cocktail: it will hinder the action of endogenous enzymes, such as proteases and phosphatases, present in crude cell extracts.

  • Lysozyme: this enzyme is also known as muramidase or N-acetylmuramide glycanhydrolase and it damages bacterial cell walls.

After a brief incubation of 30 minutes at 37 °C, immuniprecipitation buffer will be added to the mix in order to enhance the subsequent immunoprecipitation step.

• Sonication of lysate: it will break open the cells, exposing their content to the solution, and it will also shear the DNA in order to obtain smaller fragments of protein-DNA complexes.

After sonication, the mix should be centrifuged to separate the sheared protein-DNA complexes from the cell debris.