Hello ChIP-Seq community, I have pored over all the ChIP Bioanalyzer posts without finding an answer, and now I ask any advice from those of you with experience. Attached is a PDF of the Bioanalyzer results of my latest ChIP-Seq libraries, including Input, 9C/9D (tech replicates) and 27C/27D (tech replicates).
For these, one chromatin sample was crosslinked, sheared in a Bioruptor, and aliquoted into the appropriate antibody tubes.
After de-crosslinking/Prot. K, there were two methods of purification (to test the respective yields). Input, 9D and 27D were purified with 1.8 volumes of Ampure XP. Meanwhile, 9C and 27C were purified with the spin column that came with our ChIP kit. Libraries were produced in an identical manner for all DNAs using a kit, with similar amounts of starting DNA for each.
It seems to me that the "artifact" seen in 9C and 27C must arise from the column purification, but how this would translate to differences in the final library considering the adapter-ligated fragments are bead-purified before PCR? If it were a cross-linking effect, I would expect to see it in both replicates, so I assume it is a bubble product. (And therefore not terribly important, but this is driving me nuts.)
Any ideas greatly appreciated, thank you.
For these, one chromatin sample was crosslinked, sheared in a Bioruptor, and aliquoted into the appropriate antibody tubes.
After de-crosslinking/Prot. K, there were two methods of purification (to test the respective yields). Input, 9D and 27D were purified with 1.8 volumes of Ampure XP. Meanwhile, 9C and 27C were purified with the spin column that came with our ChIP kit. Libraries were produced in an identical manner for all DNAs using a kit, with similar amounts of starting DNA for each.
It seems to me that the "artifact" seen in 9C and 27C must arise from the column purification, but how this would translate to differences in the final library considering the adapter-ligated fragments are bead-purified before PCR? If it were a cross-linking effect, I would expect to see it in both replicates, so I assume it is a bubble product. (And therefore not terribly important, but this is driving me nuts.)
Any ideas greatly appreciated, thank you.
Comment