Hello Aparna Pathak

The sonication may not have worked in case of the following.

1. If the cells or nuclei are not lysed properly before or during sonication, the chromatin cannot be accessed or fragmented effectively.

2. Failure to keep the samples on ice or alternate sonication cycles with on-ice periods can allow the sample to heat up, degrading proteins and disrupting complexes.

3. Using too many cells in a single sonication batch can overwhelm the sonicator, leading to large fragments.

4. The sonicator may not have been run long enough or at a high enough intensity to shear the chromatin effectively.

5. A high concentration or long incubation with the crosslinking reagent (like formaldehyde) can make the chromatin too rigid, preventing sonication from effectively breaking it into the desired size range.

Yes, you may use MNase digestion as an alternative to sonication for fragmenting yeast chromatin for ChIP-seq. After crosslinking your yeast cells, you may apply MNase digestion to fragment the chromatin to a size suitable for ChIP-seq, typically in the 200-600 base pair range. You may need to optimize the digestion time and enzyme concentration for your specific yeast strain and growth conditions to achieve the desired fragment size.

Alternatively, you may use high-pressure homogenization, if you have access to such an equipment as it can also be used for chromatin fragmentation.

There is also another method known as CUT & RUN and CUT & Tag method. It uses tethered nucleases or transposases to cleave chromatin near a specific protein of interest, effectively fragmenting it in situ. In CUT & RUN, protein A/G-fused micrococcal nuclease (pA/G-MNase) is activated to cut DNA, while in CUT & Tag, a protein A/G-fused Tn5 transposase simultaneously cleaves and inserts sequencing adapters into the DNA. This controlled, antibody-guided fragmentation allows for high-resolution mapping of chromatin interactions in native, non-crosslinked cells.

Good Luck!