Anyone use low concentration for ChIP-seq prep?

Audry888 · 09-05-2009, 07:44 PM

Hello All,

I am having great difficulty quantitating my IP samples to use for ChIP-seq. When I used PicoGreen I found that I may only have approximately 4ng of sample. Has anyone used less than 10 ng of starting material for ChIP-seq? Any advice would help at this point!

andibody · 10-19-2009, 06:41 AM

We have prepared ChIP-Seq samples with as low as 1.5ng total amount and have obtained good results.
Make sure material is in the right size range so you can recover most of it out of the gel.

cheers

C.Griffin · 10-19-2009, 01:33 PM

Hello Andibody,

I just read your post and wanted to ask if you diluted the adaptors, and assuming so, to what degree. I have been working on generating libraries starting with low ChIP DNA input, and have some success, but measured thus far. I've managed to successfully PCR up the gel isolates, but there is evidence of what I suspect are PCR products resulting from concatamerization. I intend to isolate the 2 major PCR products by repeating a gel fractionation/extraction followed by a round of PCR, followed by, cloning verification if needed to determine which of the two species is correct.

Any thoughts?

Best regards,

Chandi

andibody · 10-29-2009, 04:33 AM

Hi,
sorry for the late answer. We diluted adaptors 1:20 but I suspect that we could even go up with the dilution. We have observed a second band double the size of the expected fragments although but we are not sure where it comes from (seems as nobody really knows yet, there are some threads on this issue). But, to my experience, this observation is not related to low starting amouts of sample.
If the bigger band is small, we add it up to quantification and sequenced all material. If it is bigger than let's say 1/3 of the 'right' band, we perform a second size selection. Both ways have given good results, nonetheless, we are still afraid of bringing any bias into our sample when this kind of artifacts show up.

best
Andi

smallcreek · 05-28-2010, 12:37 PM

Hi guys,
I am happy to read your valuable discussion here. We recently had problem that the color balance of the sequencing is completely off, which caused majority of image pannel failed. On the WFA, every parameter looks fine except P2_Gain is too low (lower than 8). Is that color balance off is caused by the concatamerization of adaptors? We did not know this trick and did not dilute the adaptors while our ChIP DNA concentration is very low.

Thanks in advance.

Smallcreek

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09-05-2009, 07:44 PM	#1
Audry888 Junior Member Location: Emeryville Join Date: Sep 2009 Posts: 1	Anyone use low concentration for ChIP-seq prep? Hello All, I am having great difficulty quantitating my IP samples to use for ChIP-seq. When I used PicoGreen I found that I may only have approximately 4ng of sample. Has anyone used less than 10 ng of starting material for ChIP-seq? Any advice would help at this point!

10-19-2009, 01:33 PM	#3
C.Griffin Junior Member Location: California Join Date: Sep 2008 Posts: 2	ChIP-seq sample prep. with low input Hello Andibody, I just read your post and wanted to ask if you diluted the adaptors, and assuming so, to what degree. I have been working on generating libraries starting with low ChIP DNA input, and have some success, but measured thus far. I've managed to successfully PCR up the gel isolates, but there is evidence of what I suspect are PCR products resulting from concatamerization. I intend to isolate the 2 major PCR products by repeating a gel fractionation/extraction followed by a round of PCR, followed by, cloning verification if needed to determine which of the two species is correct. Any thoughts? Best regards, Chandi

10-19-2009, 06:41 AM	#2
andibody Member Location: Vienna Join Date: Mar 2008 Posts: 45	We have prepared ChIP-Seq samples with as low as 1.5ng total amount and have obtained good results. Make sure material is in the right size range so you can recover most of it out of the gel. cheers

10-29-2009, 04:33 AM	#4
andibody Member Location: Vienna Join Date: Mar 2008 Posts: 45	Hi, sorry for the late answer. We diluted adaptors 1:20 but I suspect that we could even go up with the dilution. We have observed a second band double the size of the expected fragments although but we are not sure where it comes from (seems as nobody really knows yet, there are some threads on this issue). But, to my experience, this observation is not related to low starting amouts of sample. If the bigger band is small, we add it up to quantification and sequenced all material. If it is bigger than let's say 1/3 of the 'right' band, we perform a second size selection. Both ways have given good results, nonetheless, we are still afraid of bringing any bias into our sample when this kind of artifacts show up. best Andi

05-28-2010, 12:37 PM	#5
smallcreek Junior Member Location: Florida Join Date: Nov 2009 Posts: 4	Hi guys, I am happy to read your valuable discussion here. We recently had problem that the color balance of the sequencing is completely off, which caused majority of image pannel failed. On the WFA, every parameter looks fine except P2_Gain is too low (lower than 8). Is that color balance off is caused by the concatamerization of adaptors? We did not know this trick and did not dilute the adaptors while our ChIP DNA concentration is very low. Thanks in advance. Smallcreek