How can I optimize lab protocol for CHIP-Seq from rice roots?

[devbt15]

Dear CHIPers,

I have just started optimization for CHIP in my current lab since we don't have an optimized CHIP protocol in our lab. I am currently following the Abcam protocol (see attached).

A) I have started with optimizing sonication cycles. I used a Diagenode Bioruptor (with water bath) for shearing chromatin from non-crosslinked 0.4 g rice leaf powder: 3 times of 10 cycles (30 sec ON, 30 sec OFF). Please find the gel picture attached.

 

 Next will start with crosslinking and reverse-cross-linking optimization. I will perform crosslinking of the cold tissue powder instead of whole tissue and without vacuum. Thereafter proceed with nuclei extraction, lysis, and sonication immediately after this until the reverse cross-linking with NaCl at 65 degrees overnight with RNase and Proteinase K.

 

C) Finally will verify different antibody concentrations for assessing IP. I am using a monoclonal anti-FLAG antibody from Sigma, as mentioned below:

https://www.sigmaald...ng=de&region=DE

 

My questions are:

1) Do you think the gel represents the DNA fragments that I can utilize in my sequencing?

2) I would also be thankful for any tips/tricks/modifications for the steps-B and C.

 

Best regards,

D Das.

chip-using-plant-samples-arabidopsis.pdf 4Soncationeffectgel.pdf