Post PCR Enrichment Gel Selection for ChIP-Seq

Daytwa · 10-12-2011, 09:39 AM

Hello,

After I PCR enrich ChIP-seq libraries following the Illumina protocol I notice bioanalyzer peaks corresponding to what I think is primer or primer (<100bp). Has anyone else seen this? Should we consider a gel size selection post-PCR enrichment?

Thanks

HESmith · 10-12-2011, 02:28 PM

Adapter dimers are a common contaminant of Illumina libraries. If it constitutes a significant fraction of the total, I would recommend size selection after PCR enrichment. If it's only a minor contaminant, it's typically not worth the effort.

indu87 · 05-10-2012, 03:02 AM

Quote:

Originally Posted by Daytwa

Hello,

After I PCR enrich ChIP-seq libraries following the Illumina protocol I notice bioanalyzer peaks corresponding to what I think is primer or primer (<100bp). Has anyone else seen this? Should we consider a gel size selection post-PCR enrichment?

Thanks

Hi Daytwa- I notice the same with my library prep. How did you get rid of the peaks (adaptors/primers) ? Did you reduce the concentration of adaptors? Or use beads for size selection? Or gel purify again after PCR? Or run gel longer (5hrs) & slower before PCR enrichment?

Daytwa · 05-10-2012, 07:25 AM

@indu

what i've found works best (especially when constructing low input libraries (~10 ng or less) is two use the ampure xp beads following ligation (i do this 3 times). i then run a ~3% gel. Make sure you do not add EtBr to the gel. Do a post stain. It takes more time but it helps with keeping adapter aggregates from contaminating your library. Also run the gel slow. Once stained I do an old fashioned clean blade gel slice followed by a Qiagen gel purification column. After PCR enrichment I will typically do another Ampure purification. This isnt always needed but i don't notice much loss so what the hey. If you have an extra chip you can always run a Bioanalyzer high sensitivity chip to inform whether or not you need the 2nd ampure purification. hope this helps.

indu87 · 05-10-2012, 07:45 AM

thank you for your response, i think this helps. i will try beads instead of direct size selection on gel.

just one more question- i notice that the concentration of my samples are really poor in comparison to input after library prep, although the starting material used was 10ng throughout. i doubt if my initial QUBIT measurement was wrong considering that input I measured along with samples have worked fine. i re-checked my original calculations to make sure i had 10ng to begin with and this also seems ok.

what puzzles me is that i seem to have lost most of the DNA for samples and not input, whilst the original yield from the IPs were pretty good (range 100-250ng total)

any suggestions would help ! many thanks

Daytwa · 05-10-2012, 07:52 AM

@indu,

I am somewhat confused. You are worried about the concentration of you library vs the input? If that is the case don't be. It is normal for the prep to be somewhat lossy. Yields are never 100% with columns and I bet it's even worse at the gel step. That said you need so very little library to get a run that you'll likely be fine.

Also I want to stress that I do Ampure selection -> Gel -> Column Purification -> PCR ->Ampure (may or may not be needed). I would strongly recommend quantifying your library by PCR too. Also check the size distribution on a bioanalyzer if possible. These last two steps are very important to determining the quality of the library.

Best.

indu87 · 05-10-2012, 08:01 AM

hi, maybe i was not clear. pl find attached. the DNA from the CHIPs have poor yield post library prep in comparison to Input.

starting material for the library prep was 10ng for all my samples & input.

indu87 · 05-10-2012, 08:06 AM

I have got samples (after library prep) which almost look like H20 control with no peaks or very negligible sized peaks around the size range 200-300bp.
this ofcourse is after blind size selection of DNA on gel before PCR amplification.

while the input has worked very well (processed alongside samples), albeit contamination around 100bp (presumably adaptors/primers), i seem to have lost or got little DNA left from the actual IPs after library prep. the only dominating peak for my samples is the actual contaminant you can see around 100bp.

thanks for your reply in advance !

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10-12-2011, 09:39 AM	#1
Daytwa Member Location: Detroit Join Date: Dec 2010 Posts: 19	Post PCR Enrichment Gel Selection for ChIP-Seq Hello, After I PCR enrich ChIP-seq libraries following the Illumina protocol I notice bioanalyzer peaks corresponding to what I think is primer or primer (<100bp). Has anyone else seen this? Should we consider a gel size selection post-PCR enrichment? Thanks

10-12-2011, 02:28 PM	#2
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	Adapter dimers are a common contaminant of Illumina libraries. If it constitutes a significant fraction of the total, I would recommend size selection after PCR enrichment. If it's only a minor contaminant, it's typically not worth the effort.

05-10-2012, 07:25 AM	#4
Daytwa Member Location: Detroit Join Date: Dec 2010 Posts: 19	@indu what i've found works best (especially when constructing low input libraries (~10 ng or less) is two use the ampure xp beads following ligation (i do this 3 times). i then run a ~3% gel. Make sure you do not add EtBr to the gel. Do a post stain. It takes more time but it helps with keeping adapter aggregates from contaminating your library. Also run the gel slow. Once stained I do an old fashioned clean blade gel slice followed by a Qiagen gel purification column. After PCR enrichment I will typically do another Ampure purification. This isnt always needed but i don't notice much loss so what the hey. If you have an extra chip you can always run a Bioanalyzer high sensitivity chip to inform whether or not you need the 2nd ampure purification. hope this helps.

05-10-2012, 07:45 AM	#5
indu87 Junior Member Location: UK Join Date: May 2012 Posts: 4	thank you for your response, i think this helps. i will try beads instead of direct size selection on gel. just one more question- i notice that the concentration of my samples are really poor in comparison to input after library prep, although the starting material used was 10ng throughout. i doubt if my initial QUBIT measurement was wrong considering that input I measured along with samples have worked fine. i re-checked my original calculations to make sure i had 10ng to begin with and this also seems ok. what puzzles me is that i seem to have lost most of the DNA for samples and not input, whilst the original yield from the IPs were pretty good (range 100-250ng total) any suggestions would help ! many thanks

05-10-2012, 07:52 AM	#6
Daytwa Member Location: Detroit Join Date: Dec 2010 Posts: 19	@indu, I am somewhat confused. You are worried about the concentration of you library vs the input? If that is the case don't be. It is normal for the prep to be somewhat lossy. Yields are never 100% with columns and I bet it's even worse at the gel step. That said you need so very little library to get a run that you'll likely be fine. Also I want to stress that I do Ampure selection -> Gel -> Column Purification -> PCR ->Ampure (may or may not be needed). I would strongly recommend quantifying your library by PCR too. Also check the size distribution on a bioanalyzer if possible. These last two steps are very important to determining the quality of the library. Best.

05-10-2012, 08:06 AM	#8
indu87 Junior Member Location: UK Join Date: May 2012 Posts: 4	I have got samples (after library prep) which almost look like H20 control with no peaks or very negligible sized peaks around the size range 200-300bp. this ofcourse is after blind size selection of DNA on gel before PCR amplification. while the input has worked very well (processed alongside samples), albeit contamination around 100bp (presumably adaptors/primers), i seem to have lost or got little DNA left from the actual IPs after library prep. the only dominating peak for my samples is the actual contaminant you can see around 100bp. thanks for your reply in advance !