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

Sonication; Crosslinking CHIP Rice Formaldehyde

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#1 devbt15

devbt15

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Posted 20 February 2020 - 11:10 PM

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.
 
cool.png 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:
 
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.

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Also tagged with one or more of these keywords: Sonication;, Crosslinking, CHIP, Rice, Formaldehyde

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