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Old 01-09-2012, 08:49 AM   #1
Alex Clop
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Location: London

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Default Indexing ChIPseq libraries using Illumina's TruSeq and ChIPseq kits

Hi there,

I have prepared five indexed libraries from two ChIP samples and three different non immunoprecipitated chromatins (inputs) consisting in mono-nucleosomal bands (150bp long). The samples are: Sample1 H3k4me1 IP, Sample2 H3k27Ac IP, and Samples 3, 4 and 5 the 3 inputs. I must add that Sample3 is the original Chromatin that I used to for the IP in Sample1 and Sample2. I used 10 ng for the Sample2 (all the amount I had), and 20 ng for all the other samples.

I used the Illumina's standard ChIPseq kit and protocol for the end repair and the adenylation and the TruSeq DNA library prep v2 protocol for the ligation and the PCR. The only modifications relate to the TruSeq protocol and are:
(1) I dilluted the adapters in H2O to 1:40.
(2) As suggested by a colleague, I did first a 4 cycle PCR, followed by a gel size selection (cut an invisible band between 200 to 300 bp according to the ladder) and Qiagen MinElute gel purification, and a final PCR of 14 cycles. Both PCR were as suggested in the TruSeq protocol ( similar to the ChIPseq but with 60C instead of 65C annealing).

Of the 5 samples, only Sample2 worked, which is the only one that I processed with 10ng starting DNA. Sample2 showed a peak of 270 bp (150 insert size and roughly 100bp of the adapters). I run a Bionalazyer HS DNA chip and observed that two of the input chromatins have a strange ladder pattern with several peaks differing 30 bp between each other and ranging from 206 to 570 bp, which I am unable to interpret. Because I cut a band of 200-300bp I assume that this ladder has to come from the last 14 cycle-PCR but I still don’t know what could it be…concatenating primers? I have attached the bionalyzer results hoping that someone have seen that before and can give me some tips, comments, suggestions. Just for clarity, in the bioanalyzer, Sample1 to Sample5 are in the same order. The remaining traces in the bionalyzer .pdf file correspond to other samples.

Thanks to everyone.

Cheers,

Alex
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Old 01-09-2012, 09:38 AM   #2
Jon_Keats
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That is a bit weird. We have used "Ethanol"s (see users posts) protocol with great success and have not seen this issue. How are you fragmenting chromatin, and purifying the DNA?
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Old 01-11-2012, 02:14 AM   #3
Alex Clop
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Hi Jon

As I am interested in histone modifications, I am doing micrococcal digestion on native chromatin so that my major fraction correspond to mono-nucleosomal bands (150bp) with very weak di- and tri-nucleosomal fragments (300 and 450 bp respectively). After IP, I do a 2-hour RNAse treatment at 68C and a QiaQuick column purification and elution in Qiagen Elution Buffer. After quantification and qPCR to check for the ChIP enrichment comparing ChIP samples with their input counterparts, I prepare the libraries with 20 ng of DNA.

Thanks

Alex
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Old 01-11-2012, 03:37 AM   #4
Alex Clop
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Hi Jon

As I am interested in histone modifications, I am doing micrococcal digestion on native chromatin so that my major fraction correspond to mono-nucleosomal bands (150bp) with very weak di- and tri-nucleosomal fragments (300 and 450 bp respectively). After IP, I do a 2-hour RNAse treatment at 68C and a QiaQuick column purification and elution in Qiagen Elution Buffer. After quantification and qPCR to check for the ChIP enrichment comparing ChIP samples with their input counterparts, I prepare the libraries with 20 ng of DNA.

Thanks

Alex
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Old 01-22-2012, 07:42 AM   #5
Jayu
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I am interested in doing CHIPseq analysis can anyone tell me the pipeline for this.
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Old 01-23-2012, 04:17 AM   #6
pmiguel
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Quote:
Originally Posted by Alex Clop View Post
Hi there,


Of the 5 samples, only Sample2 worked, which is the only one that I processed with 10ng starting DNA. Sample2 showed a peak of 270 bp (150 insert size and roughly 100bp of the adapters). I run a Bionalazyer HS DNA chip and observed that two of the input chromatins have a strange ladder pattern with several peaks differing 30 bp between each other and ranging from 206 to 570 bp, which I am unable to interpret. Because I cut a band of 200-300bp I assume that this ladder has to come from the last 14 cycle-PCR but I still don’t know what could it be…concatenating primers?
The TruSeq protocol includes "In-line controls" -- control DNA spiked-in to various steps in the protocol. Its use is optional, but if you are using it, then that could explain the strange ladder pattern. They could be the actual control DNAs.

--
Phillip
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Old 11-08-2012, 11:07 AM   #7
dianedamezwerno
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Quote:
Originally Posted by Alex Clop View Post
Hi there,

I have prepared five indexed libraries from two ChIP samples and three different non immunoprecipitated chromatins (inputs) consisting in mono-nucleosomal bands (150bp long). The samples are: Sample1 H3k4me1 IP, Sample2 H3k27Ac IP, and Samples 3, 4 and 5 the 3 inputs. I must add that Sample3 is the original Chromatin that I used to for the IP in Sample1 and Sample2. I used 10 ng for the Sample2 (all the amount I had), and 20 ng for all the other samples.

I used the Illumina's standard ChIPseq kit and protocol for the end repair and the adenylation and the TruSeq DNA library prep v2 protocol for the ligation and the PCR. The only modifications relate to the TruSeq protocol and are:
(1) I dilluted the adapters in H2O to 1:40.
(2) As suggested by a colleague, I did first a 4 cycle PCR, followed by a gel size selection (cut an invisible band between 200 to 300 bp according to the ladder) and Qiagen MinElute gel purification, and a final PCR of 14 cycles. Both PCR were as suggested in the TruSeq protocol ( similar to the ChIPseq but with 60C instead of 65C annealing).

Of the 5 samples, only Sample2 worked, which is the only one that I processed with 10ng starting DNA. Sample2 showed a peak of 270 bp (150 insert size and roughly 100bp of the adapters). I run a Bionalazyer HS DNA chip and observed that two of the input chromatins have a strange ladder pattern with several peaks differing 30 bp between each other and ranging from 206 to 570 bp, which I am unable to interpret. Because I cut a band of 200-300bp I assume that this ladder has to come from the last 14 cycle-PCR but I still don’t know what could it be…concatenating primers? I have attached the bionalyzer results hoping that someone have seen that before and can give me some tips, comments, suggestions. Just for clarity, in the bioanalyzer, Sample1 to Sample5 are in the same order. The remaining traces in the bionalyzer .pdf file correspond to other samples.

Thanks to everyone.

Cheers,

Alex
Hi Alex ,

I am very curious to find out your conclusions about "this several peaks differing 30 bp between each other and ranging from 206 to 570 bp " bioanalyzer profile as I have been running in the exact same problem for only SOME of my libraries.
And not only once!
I have no explanation and it is pretty stressful.

Any suggestion would be GREATLY appreciated!
Looking forward hearing from you.
Sincerely.
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