Hi,

I had the same issues with fibroblasts and other adherent cells. I tested different lysis buffers and procedures (e.g. douncer) to isolate nice nuclei from fixed cells but it never worked out. Compared to other cell types or tissues you will always find a lot of cytoplasm still attached to the nuclei in these cells. This remaining cytoplasm causes the aggregation of cells after you added SDS.

But 4C-seq also works with these cells. I recommend the following to prevent the massive aggregation of cells:

1. reduce the number of cells in your reaction. I use 2.5 million cells per reaction.

2. add the SDS sequentially and try to constantly disaggregate clumps carefully by pipetting during SDS step.

3. after quenching with triton, mix again by pipetting to disaggregate the clumps. In subsequent digestion steps you will always find smaller remaining aggregates but they don’t interfere with digestion efficiency or ligation.

Good luck for your experiments.

Best,

Martin

Hi Martin 

Thanks for your suggestions and good explanation. Actually I ave used the strangest lyse buffer but I didn't check the quality of lysing by staining technique.

I will try to check the lyse efficiency.

I am using HindIII as first enzyme and one four cutter for second one.

Do you have any slide of gel control of first digestion to send to me? I want to compare with mine.

Thanks

Reza