SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Best device for chromatin shearing/ChIP-seq? eab Epigenetics 0 07-19-2018 12:22 PM
Covaris S2: Chromatin shearing for ChIP(-seq?) aleidenroth Sample Prep / Library Generation 8 01-16-2017 07:06 AM
Chromatin shearing for ChIP-Seq using Covaris S2 menkelab Sample Prep / Library Generation 0 03-24-2010 10:10 AM
Chromatin shearing for ChIP-Seq using Covaris S2 menkelab Illumina/Solexa 0 03-24-2010 04:57 AM

Reply
 
Thread Tools
Old 12-09-2019, 05:35 AM   #1
Shahrukh
Junior Member
 
Location: Russia

Join Date: Oct 2019
Posts: 3
Post Shearing chromatin - ChIP-seq for H3K9me3

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.
Attached Files
File Type: pdf ChIP-seq_Protocol_v011014.pdf (1.76 MB, 1 views)

Last edited by Shahrukh; 12-15-2019 at 05:51 AM.
Shahrukh is offline   Reply With Quote
Old 12-13-2019, 02:25 AM   #2
Shahrukh
Junior Member
 
Location: Russia

Join Date: Oct 2019
Posts: 3
Default

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.
Attached Images
File Type: jpeg Sonication.jpeg (23.8 KB, 7 views)

Last edited by Shahrukh; 12-13-2019 at 02:36 AM.
Shahrukh is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:44 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO