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I've been trying to shear formaldehyde-crosslinked chromatin using a Branson S250 for ChIP-Seq, and I've been having some problems with attaining the correct size distribution.
I tried reading all of the posts related to shearing, so if someone could point me in the right direction, that would be great.
Basically, I keep getting a bimodal size distribution with a cluster at ~2.5 kb that is really recalcitrant to being sheared. I can break it up with longer shearing times, but at this point my fragments are too small (mostly <300 bp). I heard that one should have a tight size distribution (200-800 bp) for going on to the ChIP and library prep.
I've attached a shearing time course using Bioanalyzer.
Here are the details:
I've been following a procedure from a 2006 Nature Methods paper from Richard Young's group for the crosslinking (10 minutes in media with 1% methanol-free formaldehyde at room temp.) and lysing conditions (nuclear isolation).
The shearing conditions are:
1. 15 million cells in ~1 mL of sonication buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.5% N-laurylsarkosyl, 0.1% Deoxycholate).
2. 45% amplitude, 0.7 s pulse on, 1.3 pulse off, then timecourse.
I tried using SDS (0.1% or 0.2%) instead of N-laurylsarkosyl with not much success. I was thinking about going to 1%.
Any help would be greatly appreciated!!!
I tried reading all of the posts related to shearing, so if someone could point me in the right direction, that would be great.
Basically, I keep getting a bimodal size distribution with a cluster at ~2.5 kb that is really recalcitrant to being sheared. I can break it up with longer shearing times, but at this point my fragments are too small (mostly <300 bp). I heard that one should have a tight size distribution (200-800 bp) for going on to the ChIP and library prep.
I've attached a shearing time course using Bioanalyzer.
Here are the details:
I've been following a procedure from a 2006 Nature Methods paper from Richard Young's group for the crosslinking (10 minutes in media with 1% methanol-free formaldehyde at room temp.) and lysing conditions (nuclear isolation).
The shearing conditions are:
1. 15 million cells in ~1 mL of sonication buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.5% N-laurylsarkosyl, 0.1% Deoxycholate).
2. 45% amplitude, 0.7 s pulse on, 1.3 pulse off, then timecourse.
I tried using SDS (0.1% or 0.2%) instead of N-laurylsarkosyl with not much success. I was thinking about going to 1%.
Any help would be greatly appreciated!!!
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