Opinion: Skip Size Selection Step?

jazz710 · 07-14-2014, 09:22 AM

Hey everyone. I looked through the forums and this topic hadn't been mentioned in a while, so I hope it's alright to bring it back up.

I'm going to run a ChIP-seq experiment and this will be the first time I'm trying to make my own libraries (Illumina TruSeq ChIP-Seq Kit).

My chromatin was sheared via enzymatic digestion using the Atlantis dsDNAse from Zymo. The chromtain was sheared down to mononucleosomal level (see attached gel picture (1kb+ Ladder, Sample 1, Sample 2, Sample 3).

My question is this: If my DNA is already sheared down to mononucleosomal levels, I can skip the size selection step for the library prep, right? I'm capable of running gels and excising bands, but I have 48 libraries to prep so I'd really rather not do more work if it's not helpful. Thoughts?

nucacidhunter · 07-14-2014, 05:10 PM

Gel excision step in Illumina’s ChIPSeq protocol is to remove unligated adapters and adapters ligated to other ones (this can only happen in absence of A overhang!). Adapter dimers are around 120 bp after PCR and adapter multimers obviously will be longer. Illumina recommends gel cut in 280-320 bp range to ensure that adapters have been removed and they will not end up in sequencing library. My experience is that purification of ligation products with 1x beads twice is sufficient to remove remaining adapters and it will select the insert sizes larger than 50 bp which is recommended cycle number for ChIPseq. Larger fragments are not a problem as they will be selected against during clustering. So, my suggestion would be to skip size-selection, but to be sure that no adapter residues are carried over perform a small scale PCR (15-20 ul) with couple of samples and run the product straight on Bioanalyser or TapeStation to asses presence of primer dimers. If they look OK , then you can proceed for full scale PCR for all samples.

jazz710 · 07-15-2014, 01:08 PM

Quote:

Originally Posted by nucacidhunter

So, my suggestion would be to skip size-selection, but to be sure that no adapter residues are carried over perform a small scale PCR (15-20 ul) with couple of samples and run the product straight on Bioanalyser or TapeStation to asses presence of primer dimers. If they look OK , then you can proceed for full scale PCR for all samples.

Appreciate the advice! I'll still humor other responses, but that was my feeling as well.

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07-14-2014, 05:10 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Gel excision step in Illumina’s ChIPSeq protocol is to remove unligated adapters and adapters ligated to other ones (this can only happen in absence of A overhang!). Adapter dimers are around 120 bp after PCR and adapter multimers obviously will be longer. Illumina recommends gel cut in 280-320 bp range to ensure that adapters have been removed and they will not end up in sequencing library. My experience is that purification of ligation products with 1x beads twice is sufficient to remove remaining adapters and it will select the insert sizes larger than 50 bp which is recommended cycle number for ChIPseq. Larger fragments are not a problem as they will be selected against during clustering. So, my suggestion would be to skip size-selection, but to be sure that no adapter residues are carried over perform a small scale PCR (15-20 ul) with couple of samples and run the product straight on Bioanalyser or TapeStation to asses presence of primer dimers. If they look OK , then you can proceed for full scale PCR for all samples.