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Some good comments on ChIP-seq library prep

April 9, 2013

Well I suspected the Illumina ChIP-seq kit wasn’t very good.  Well here is what someone else had to say about it and some other good comments on making ChIP-seq library preps.

Thanks Luke!!!

Ethan,

Just thought I would let you know my experiences with the new TruSeq kit after our discussions. I have spent the last week or so playing with it. Everything now works great but there are few issues with the TruSeq ChIP kit and so here are my experiences:

 

1)      The SYBR Gold gel protocol is madness!!! This dye, when added in to the gel (as opposed to post-gel staining) completely and utterly screws up the way the gel runs. I should have known this, but have always used EtBr and so don’t know an awful lot about the SYBR stains. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX I’m amazed they ever got it to work. It’s impossible to size select accurately in my hands.

2)      Your pre-gel PCR is pure genius. I do 5 cycles as you suggest and it works a treat. I can usually see a faint smear from about 300 to 700 bp. I have tried both Phusion and Kapa and I agree that Kapa has the edge.

3)      There is minimal danger of selecting out mono DNA after ligation using the Ampure XP beads in my hands. When you think about it, a 1:1 ratio of beads to sample comfortably leaves anything over 200 bp and adapted mono-DNA is closer to 300 bp at that point. The lowest MW DNA I can see on the gel is a shade under 300 bp, which corresponds nicely to mono-DNA. Nevertheless I may add additional PEG as you suggest at this stage just in case.

4)      I only do a single post-ligation Ampure XP clean-up – primarily for speed – and it works fine, but also to reduce the risk of losing LMW DNA. I have never seen adapter contamination using this approach, at least when combined with pre-gel PCR.

5)      I use Qiagen for all steps except post-ligation and post-enrichment (for cleanliness). Qiagen post-ligation left a ton of adapters in the final library and is clearly a no-no. This increases speed with little effect on yield.

6)      You cannot dilute the Illumina indexes. I only tested 1:10 and 1:50 dilutions and got no library at all in the 1:50 and barely anything in the 1:10. It’s feasible that a 1:2 to 1:5 dilution could work, but this is unnecessary really given the quality of the final library using neat indexes.

7)      The number of enrichment cycles varies but for safety I find around 10 post-gel cycles gives a very nice amount of library at around 10- 15 ng/ul when starting out with less than 10 ng. I have gone as low as 8 cycles and it usually works fine, but the final concentration can get a bit low. I can usually tell how many I will need depending on if I can see anything on the gel or not.

 

Hope this helps in case anyone else asks your advice on this. Thanks for your tips.

 

Luke

Luke Norton, Ph.D.

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9 Comments
  1. Brian Johnson permalink

    Boy, I really wish I had stumbled upon this post a few weeks earlier when I started using the Illumina TruSeq ChIP Kit (with no prior experience with ChIP-seq prep). I just finished a practice prep earlier today using the kit on several 10ng aliquots of input DNA along with a few minor adjustments (after several failed attempts). Just for the sake of consensus:

    In reference to point #1 & 2, I also had the same trouble with the SYBR Gold pre-stained agarose gels. In my hands, different amounts of the same ladder would migrate at slightly different speeds (if they migrated properly to begin with). Doing the pre-gel 5-cycle PCR, and then running the product out on a 2.5% gel with no DNA stain (followed by a 30-minute incubation in 1X SYBR Gold) completely resolved that issue. After post-gel PCR enrichment, the bands were exactly where I expected to see them on an agarose gel. And yes, I also saw a faint smear on the gel, which just made me feel confident that the prep was working.

    And I completely agree with point #6. Diluting the TruSeq ChIP adapter indexes is a big no-no. For me, diluting it 1:10 reduced the final yield of the PCR-enriched library roughly 5-fold (again, starting from 10ng of input DNA). I am curious though as to whether diluting the indexes 1:10 would work for lower amounts of starting material (e.g. 1ng input DNA).

    For comparison, I also prepared a library in parallel using Ethan’s TruSeq ChIP protocol (but using NEBNext End Repair and A-tailing modules for more simplicity, as well as Illumina’s TruSeq Adapter Indexes at the recommended 2.5ul volume). For the library prepped according to Illumina’s protocol, I used the Primer Mix and PCR cocktail provided in the kit. With the library according to Ethan’s protocol, I used the Kapa HiFi PCR Mix along with homemade primers. I have to wait for the Bioanalyzer results to come in, but following a 10-cycle post-gel PCR enrichment, I got 9ng/ul with the Illumina library and a surprising 45ng/ul with the other by Qubit quantification (all in ~18ul total volume). I’m not sure if that’s due to a combination of both the NEB reagents and the Kapa HiFi Mix, but the reagents are cheap and simple enough (and the Illumina TruSeq ChIP indexes bountiful enough) that I’ll just stick to that protocol.

    I’m assuming this is fairly obvious and probably standard practice, but I should also recommend that one should try and measure the concentration of their DNA prior to beginning the library prep, especially if it’s been stored in the -20C for a while, rather than assume that the concentration will stay the same. A minor oversight that set me back quite a bit.

    I do have a question though: For determining the ideal PCR enrichment cycles by qPCR, you mention in your protocol that you need to determine the number of cycles need to get 50% of amplification. Is that simply just the Ct value that’s reported for each individual sample, or is there some more complicated process involved that I’m not aware of?

    -Brian

  2. ethanomics permalink

    Brian – Thanks for sharing!!!!! It’s great to hear what other people are doing!!! And sorry for the delay in getting your comments up. The flu, babies, experiments, etc. and I just kind of forgot.

    As far as cycle determination, that is just the Ct value for each individual sample at 50% amplification (non-logrithmic, i.e. about a cycle from the end-point). Depending on software capabilities you can just eyeball it.

    Ethan

  3. ethanomics permalink

    And some good recommendations from my my old frienamy Cold Spring Harbor:

    Hey Ethan

    I was just reading your blog, you posted some comments from a Truseq ChIP-seq kit user.
    The person mentioned that you can not dilute the adaptor indexes.

    The adaptor indexes in the ChIP-seq library prep kit is more diluted than the indexes in the genomic DNA libraries prep kit, so in our lab we order the kit for genomic DNA, and dilute the indexes for 50-100 times and use it for ChIP-seq library construction (and use NEB enzyme, Kapa polymerase etc like you suggested). We really just want their adaptors…and it’s much cheaper this way than ordering the ChIP-seq Kit.

    Just something to share with ChIP-seq library makers. 🙂

    Ann

    Thanks Ann!!!

  4. Luke Norton permalink

    I must say that I have become very unhappy with the reproducibility of the TruSeq kit, and my preps have started to fail consistently, most likely due to degradation of either the end-repair and/or the a-tailing mix. This was probably caused by too many freeze thaws on my part as the Illumina reagents come as ready-to-go mixes, but it could easily be due to something else going bad in the kit. In any case, I am now routinely using the Kapa library prep kit with the Illumina adapters and primer cocktail and am much happier with it. I can lower the overall number of PCR cycles by at least 3 and the consistency is great.

  5. ethanomics permalink

    I prepared some methylC-seq libraries with the TruSeq DNA kit and they probably turned out o.k. But I re-did them using End-It and NEB Klenow exo- and I got 4x more DNA after PCR. Seems like the DNA kit is fine for less sensitive libraries where you have a lot of DNA to lose, but more difficult libraries like ChIP-seq or methylC-seq, it’s not very good. And as you point out the convenience of master mixes may be at the price of enzyme activity where some is lost after each freeze-thaw cycle.

  6. ethanomics permalink

    I edited Luke’s comments as I’ve been told by our Illumina contact that it’s not nice to call people names. I totally agree and outside of politics facts make a better argument. But, yes it is beyond frustrating to buy a kit or product for my research and then feel like I’m doing the trouble shooting that should have been done before the kit was released or just wasted a chunk of our research budget on a plane shoddy product.

    I haven’t tried Illumina’s ChIP-seq library kit. It simply took too long to come. It was well over a year past the release of the TruSeq technology. I’d like to think my protocol is better but I don’t know for sure. It would have been nice if Illumina sent me an e-mail when I applied for those jobs a while back. I bet they would have ended up with a better ChIP-seq kit. I’m not sure why they didn’t just copy my protocol like Bioo Scientific did. People like that kit.

  7. Thanks all of you! Now I’m doing MethylCap-Seq and ChIP-Seq in the same time and I do facing lots of problem both in bioinformatics and wet lab base. Really appreciate all the comments and posts. Just little thing I can share, I use AMPure XP beads for size selection and NEBNext ChIP-Seq lib prep kit but with Illumina PCR Master Mix and Primer Premix Cocktail last enrichment step. Luckily my index adaptors from Illumina DNA lib prep kit and we diluted 1:10 to use, sounds good for us.
    10ng ChIP input DNA went into library prep, for PCR enrichment step, 12 cycles using but I got 20% duplication rate, so definitely need to reduce the PCR cycles.
    For cluster density determination, I will use KAPA quant kit.
    Thanks again for the sharing 😀

  8. Alex Clop permalink

    Hi Ethan and bloggers!

    I have some tissues (pig uterus and ovaries) that I kept at -80C for now over 7 years and which I would like to use for Native ChIPseq for histone modifications. However I am not 100% confident whether these samples will be suitable for this experiment. Any comments.

    Apologies for posting this question as a reply to this threat and also if this site is not aimed at recieving these questions!

    Thanks

    Alex

  9. The dude permalink

    hey dude
    do not run your gel prestained with the SYBR dye, or even load your samples with the dye. Stain it later in a 0,5xTBE buffer wit 1/50000 or 1/5000 dilution of SYBR die. That’s the trick.

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