How do I isolate nuclei before my ChIP experiment?

Conventional methods of nuclei isolation include chemical treatments with hypotonic buffers. Optimizing the duration of lysis in the buffer is crucial for nuclei isolation. The goal is to achieve thorough lysis, preferably leaving less than 5% of live cells remaining in the suspension afterward. Although single nuclei isolation essentially disrupts whole cells, it's essential to start with a high-quality sample of viable cells to ensure successful lysis. Furthermore, it's important to ensure that the isolated nuclei maintain their rounded shape and intact nuclear membrane. Prolonged lysis can cause the nucleus to bleb, resulting in a bubbled surface,  potential clumping with other nuclei and an abnormal shape.

A novel method (termed NEXSON) has been established to extract nuclei from fixed cells without relying on chemical treatments or mechanical homogenization. Instead, ultrasound is utilized to lyse the cell membrane. The process involves two steps: resuspending the cell pellet in a buffer compatible with nuclei extraction and subjecting it to moderate sonication. The buffer must ensure the nuclei are intact and should not contain ionic detergents that could disrupt the nuclear membrane. Nuclei extraction is monitored visually utilizing a benchtop phase-contrast microscope. Nuclei are counted at specific time points, and sonication is terminated when a sufficient number of nuclei has been extracted (over 70%). Adjustments in sonication time, varying between cell types (30 sec–5 min) and fixation conditions, are made in real-time based on microscope observation of the target specimen.