Dear all,
I want to do a ChIP-seq experiment using a H3K4me1 antibody and have put in place a native chromatin extraction protocol with micrococcal nuclease digestion. With this protocol one ends up with three tubes each containing a different chromatin fraction: S1 (mostly mono-nucleosomes), S2 (mono- di- tri- tetra- and even penta- nucleosomes), P (debris with some residual chromatin).
My concern (as stated in another thread some weeks ago) relates to the fragment size and range ideal for the Illumina Sequencing. I did a digestion timecourse with the micrococcal nuclease and run the gel that you can see in the attachment. I used 2 different micrococcal amounts (10 and 30 Units) and 4 different digestion times (10, 15, 20, 30 minutes). For all the 8 conditions, the nanodrop measurements indicated similar yields of chromatin.
Looking at that gel, I would say that for the ChIP-seq experiment, the best condition is 30 U of enzyme and 30 minutes digestion which shows mostly mono- and di- and some tri- nucleosomes.
Am I right in saying this? Any comment or suggestion from anyone?
Thank you in advance once more.
Alex
I want to do a ChIP-seq experiment using a H3K4me1 antibody and have put in place a native chromatin extraction protocol with micrococcal nuclease digestion. With this protocol one ends up with three tubes each containing a different chromatin fraction: S1 (mostly mono-nucleosomes), S2 (mono- di- tri- tetra- and even penta- nucleosomes), P (debris with some residual chromatin).
My concern (as stated in another thread some weeks ago) relates to the fragment size and range ideal for the Illumina Sequencing. I did a digestion timecourse with the micrococcal nuclease and run the gel that you can see in the attachment. I used 2 different micrococcal amounts (10 and 30 Units) and 4 different digestion times (10, 15, 20, 30 minutes). For all the 8 conditions, the nanodrop measurements indicated similar yields of chromatin.
Looking at that gel, I would say that for the ChIP-seq experiment, the best condition is 30 U of enzyme and 30 minutes digestion which shows mostly mono- and di- and some tri- nucleosomes.
Am I right in saying this? Any comment or suggestion from anyone?
Thank you in advance once more.
Alex