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H3K27ac ChIP - Why do I get mostly large fragments IP'ed shown by Bioanalyzer pre lib
Hi all. I think I've read all the old similar threads, but none of them had a definitive solution and I'm still stuck with this issue. So...
I'm doing H3K27ac ChIP from mouse Bone marrow derived macrophages. Although fragment length analyzed by agarose gel seem fine after shearing (enrichment from 200bp-600bp, which also seem consistent by bioanalyzer), my actual ChIP'ed samples seem to be very enriched for larger fragments (2-10kb) after bioanalyzer analysis (pre library prep), despite these fragments represent the minority of the pool on input (as show by pmol/l). The final goal is to do a ChIP-seq, but I'm concerned this issue may ruin the experiment.
By qPCR I get a very good enrichment of constitutive H3K27ac regions on housekeeping promoters. My concern is actually that I want to see a clear induction after LPS treatment, and I do see it for a few targets, but not for other very important target that I know should be enriched by looking at other available public ChIPseq data.
To make things worse, a previous postdoc had made this experiment before and we actually got to the point of sequencing her samples. We can see constitutive H3K27ac enrichment at expected regions (housekeeping promoters) as I see in my experiments by qPCR, BUT we don’t see an LPS induction at expected target genes at all, which is what I’m trying to troubleshoot now. It’s worth mentioning that her bioanalyzer results before library prep showed the same pattern of large fragments ChIP’ed, hence why I’m paranoid with that.
I'm sending some more detailed information about the experiment and attaching gel/bioanalyzer results. Any thoughts about the issue would be really appreciated.
- Crosslinked for 8’ with 1% fresh paraformaldehyde ampoule; quenched with glycine 0.125M for 10’
- Did 2 experiments, one using 15 million cells, another with 5 million cells per IP in 300uL of RIPA buffer (10mM Tris, 1mM EDTA, 140mM NaCl, 1% Triton, 0.1% SDS, 0.1% Na-DOC) sonicated with Bioruptor Twin – I did multiple tests but went with 3 rounds of 10 cycles 30” ON/OFF
- Incubated the whole sonicated extract with 30uL of dynabeads pre-coated with 5ug of H3K27ac antibody at 4oC O.N
- Followed the rest of standard washing and decrosslink, purified DNA with QIAgen colums.
I'm doing H3K27ac ChIP from mouse Bone marrow derived macrophages. Although fragment length analyzed by agarose gel seem fine after shearing (enrichment from 200bp-600bp, which also seem consistent by bioanalyzer), my actual ChIP'ed samples seem to be very enriched for larger fragments (2-10kb) after bioanalyzer analysis (pre library prep), despite these fragments represent the minority of the pool on input (as show by pmol/l). The final goal is to do a ChIP-seq, but I'm concerned this issue may ruin the experiment.
By qPCR I get a very good enrichment of constitutive H3K27ac regions on housekeeping promoters. My concern is actually that I want to see a clear induction after LPS treatment, and I do see it for a few targets, but not for other very important target that I know should be enriched by looking at other available public ChIPseq data.
To make things worse, a previous postdoc had made this experiment before and we actually got to the point of sequencing her samples. We can see constitutive H3K27ac enrichment at expected regions (housekeeping promoters) as I see in my experiments by qPCR, BUT we don’t see an LPS induction at expected target genes at all, which is what I’m trying to troubleshoot now. It’s worth mentioning that her bioanalyzer results before library prep showed the same pattern of large fragments ChIP’ed, hence why I’m paranoid with that.
I'm sending some more detailed information about the experiment and attaching gel/bioanalyzer results. Any thoughts about the issue would be really appreciated.
- Crosslinked for 8’ with 1% fresh paraformaldehyde ampoule; quenched with glycine 0.125M for 10’
- Did 2 experiments, one using 15 million cells, another with 5 million cells per IP in 300uL of RIPA buffer (10mM Tris, 1mM EDTA, 140mM NaCl, 1% Triton, 0.1% SDS, 0.1% Na-DOC) sonicated with Bioruptor Twin – I did multiple tests but went with 3 rounds of 10 cycles 30” ON/OFF
- Incubated the whole sonicated extract with 30uL of dynabeads pre-coated with 5ug of H3K27ac antibody at 4oC O.N
- Followed the rest of standard washing and decrosslink, purified DNA with QIAgen colums.