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-   -   Shearing chromatin - ChIP-seq for H3K9me3 (http://seqanswers.com/forums/showthread.php?t=92016)

Shahrukh 12-09-2019 06:35 AM

Shearing chromatin - ChIP-seq for H3K9me3
 
1 Attachment(s)
Hi everyone,
It's my first time posting on seqanswers.
I am shearing chromatin of HEK293T ~5m cells. I am using the ChIP-seq protocol from Myers lab/Encode consortia. Looking at 200-600bp sheared chromatin.
We have a Sonics Vibra cell sonicator in our lab. It has the smallest microtip as 1/8 tapered microtip. I want to do some trials before performing it for my treated samples. If anyone has worked with this type of sonicator before kindly share your experience and the protocol you followed.
I am specifically interested in knowing:

1. The amplitude?
2. The number of cycles?
3. The time?

There is a lot of information regarding other sonicators but not compatible with the one we have. I would be grateful if someone can shed light on this. Thanks in advance :)

I am attaching here the ChIP-seq protocol I am using.

Shahrukh 12-13-2019 03:25 AM

1 Attachment(s)
So this week, I tried sonication with Sonics Vibra cell 500/750. I want to share my experience and hopefully get input from you. The details are as follows:
- ~3m HEK293T cells per sample
- 500ul RIPA buffer per sample
- 1/8" tapered microtip from Sonics
The sonication conditions I used on four technical replicates are:
A = 3 sec on, 30 sec off at 30%amp for 10 min
B = 3 sec on, 30 sec off at 30%amp for 20 min
C = 3 sec on, 30 sec off at 20%amp for 20 min
D = 3 sec on, 30 sec off at 25%amp for 20 min

All of them had foaming except for sample C, which was sonicated at 20% amplitude. DNA was extracted by using a local Russian kit. I got the following amounts for DNA fragments without being IP'd.
A = 0.33ug
B = 0.32ug
C = 0.38ug
D = 0.26ug
Attached is the gel (sorry our gel doc broken camera is broken). I believe I can play a bit more with the conditions described in C by decreasing the total time to 15 and 10min. Please let me know what'd be suitable.
Also, what's with the low conc.? I didn't even IP'd them. Is it due to less number of cells? Any suggestion to increase the DNA amount.


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