Problems with ChIP-seq library construction

[zwang0614]

Hi all.
I am trying to prepare several ChIP-seq libraries. I started with 3ng DNA, which was sonicated to 100-500bp length. After the blunt ending, A tail adding, and adapter ligation, I did the qPCR to do the quantitation but found most of the qPCR products were adaptor dimers (about 120bp). Does anybody have similar experiences or any suggestions? Thanks.

Z.

[chen]

Hi zwang,

we had a similar result starting with ChIP-DNA, using the Truseq nano pre kit. And we find some smear above the dimers when we redo the PCR step, adding the cycles up to 30 comparing with protocol's 8 cycle. we gel purify the PCR product.

but we are not quite sure about the smear.
May someone give any advice.

[NextGenSeq]

30 cycles will lead to a lot of duplicate reads. We typically do 13 to 17 depending on the starting amount of ChIP-DNA.

Reduce the amount of adapters in the ligation step when you start with small amounts of ChIP-DNA. This helps reduce adapter artifacts.

[chen]

30 cycles will lead to a lot of duplicate reads. We typically do 13 to 17 depending on the starting amount of ChIP-DNA.

Reduce the amount of adapters in the ligation step when you start with small amounts of ChIP-DNA. This helps reduce adapter artifacts.

yeah. yet this was our first try ChIP-seq library, so we just stick to the protocol(Truseq Nano DNA kit). we may improve the ligation step next time.

we would take the smear library to do a test run. Hope the data would be not so bad.

[NextGenSeq]

I personally do not like the Illumina Nano kit. We get better results with the NuGen Ultralow or Diagenode ChIP kit.