Strange higher size amplicon in ChIP seq Library!!

[Chiper]

Hi, I am seeing a strange amplicon ~2Kb long in all my libraries beside expected ~250-300bp amplicon (picture attached).

I perform ChIP using DNA sheared with bioruptor which peaks ~500bp. I work with low cell number so I get ~5-10 ng after ChIP. Post IP this DNA is further sheared using Covaris and processed for library preparation as per the Solid ChIP-seq library preparation protocol. I used to get ~250-300 bp DNA as expected but in all my recent samples I could see a higher size amplicon ~2 KB beside the expected amplicon. I am not able to find any logical explanation for this!! Has anyone seen this before?

Can I still use these libraries for sequencing after size selection? I am afraid I might introduce bias in my samples. Please help.

[Hamid]

Hi Chiper,

what reverse cross linking method are you using?
How are you purifying the IP'd material for subsequent fragmentation on the Covaris instrument?


Thank you

Hamid

[Chiper]

Dear Hamid,
Thanks for your questions. I heat the ptn-DNA complex at 67 degrees for 4-5 hours to reverse the cross links. After Ptn-K treatment DNA from ChIP is purified using Qiagen PCR Purification kit and qPCR is done to ensure that ChIP has worked fine.

After all these steps IPed DNA (~40-45 ul) is diluted in low TE to get 100 ul final volume and further sonicated using Covaris to get smaller size fragments (~100 bp). Since DNA amount is low we don't do any size selection at this step. All the subsequent purification of DNA are done with the help of Agencourt magnetic beads as per the standard protocol. If we are leaving some population un-sheared it's puzzling to me that we are seeing a sharp peak around 2Kb. Please let me know if you need more information.

[zwang0614]

It is really weird. Has it been solved?

[NextGenSeq]

The Bioruptor sucks for shearing. I would switch to the Covaris.

[AndersHaakon]

The Bioruptor sucks for shearing. I would switch to the Covaris.

I've heard this a few time now here - and frankly we are having quite a lot of problems with our BioRuptor on chromatin prepared from some cell types.

Did you do a direct comparison between BioRuptor and Covaris, and was the comparison done on material that you were unable to shear with the bioruptor?

Thx in advance,
Anders