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Old 04-29-2012, 12:20 PM   #1
lyxsdu2006
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Default Can I prepare ChIP-seq library by using TruSeq DNA sample preparation kit

Hi,

We just got Miseq in our lab. And we are going to sequence ChIP'ed DNA in bacteria and bacterial genome using Miseq. We purchased illumina Truseq DNA sample preparation kit to construct libraries for both ChIP'ed DNA and genomic DNA. So I am wondering if anyone used Truseq DNA sample preparation kit to construct ChIP'ed DNA library. And did it work well?

Thank you!
Flora
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Old 04-30-2012, 10:27 AM   #2
jlove
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Hi Flora,
We do almost exclusively ChIP-Seq in our facility, using the TruSeq kit.
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Old 04-30-2012, 02:58 PM   #3
lterhune
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Hi jlove,

We have also been interested in trying ChIP-seq with the TruSeq kit-- could you tell me what changes you made to the original TruSeq protocol (like diluting adapters or other enzymes, etc)?

We found this protocol but haven't tried it yet (from Ethanol I believe): http://ethanomics.wordpress.com/chip...useq-adapters/

Is your procedure similar to the above protocol?
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Old 05-01-2012, 08:37 AM   #4
lyxsdu2006
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Hi jlove,

Thank you for your reply. I am also wondering if you did any changes by using original Trueseq kit. We also found the protocol from Ethan, but the problem is that everthing provided by illumina is mixed already(end repair mix, A-Tailing mix, ect). Illmina doesn't provide seperate enzymes as what is mentioned in Ethan's protocol.So it is hard for us to scale down. And we did our library construction by using Trueseq kit and just finished our first round sequencing, but the result is not good. Most of reads are self-ligation of adapters. What do you think the problems are and could you please tell me your changes by using TruSeq kit?

Thank you so much!
Flora
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Old 05-01-2012, 09:17 AM   #5
TonyBrooks
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The main thing to consider is the dilution of the adapter. I believe the TruSeq adapters are at 15ÁM and you add 2.5ÁL.
Most ChIP protocols I've seen use a 1.5ÁM adapter and add 1ÁL (starting with 10ng ChIP DNA). Too much adapter will promote adapter-adapter ligation.
Did you QC the libraries on the Bioanalyser? If so, did you see the adapter-dimer peak?
You could always gel-cut the final library which will reduce the dimers.
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Old 05-01-2012, 12:35 PM   #6
Jon_Keats
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We use the protocol from ETHANol and it works great. I've thought of buying a TruSeq kit for the adaptors (then diluting 1/10 or 1/100 most likely) and PPC oligos only but after buying oligos from IDT I have enough for >4000 libraries for each of the 12 barcodes so maybe in my next life..
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Old 05-01-2012, 01:40 PM   #7
msheldon
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Hi Flora,
We're using the Bioo ChipSeq kit and barcodes with great results. Their adapters are the long type mentioned in this thread. The price is pretty good too. I talked to them on the phone and asked how they did it, and they said they used lower concentrated adapters with modifications to the enzymes. They added things to the buffers like PEG to increase the probability of insert hitting adapter.
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Old 05-08-2012, 09:04 AM   #8
jlove
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I use the TruSeq adapters at 1:50 for any ChIP input from 5-50 ng and do 18 cycles of PCR before size selection.
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Old 05-08-2012, 07:41 PM   #9
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Our attempts with TruSeq and diluting adapters has so far been unsuccessful. But we have used the Bioo Chip-Seq kit with success. Their Chip adapters are more dilute than their regular DNA adapters.
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