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ChIP-seq library construction using the Illumina TruSeq Adapters

A few more updates to the protocol.  This version is working really well.  I have increased the PEG concentration used with the AMPure beads to increase the recovery of the smallest DNA fragments.  I have also reduced the enzymes used some The 30˚C incubation with ligase seemed to produce a lot of adapter dimer, so I got rid of that.  Illumina is using Indexes 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18 and 19 in their kits now so I would go for those first.  I have also uploaded a most likely copyrighted pdf with 48 TruSeq indexes (this is information that Illumina users need to know so I would guess they wouldn’t care).

ChIP-seq library construction using the Illumina TruSeq Adapters

Adapter Index Key

11 Comments
  1. gillt permalink

    Hello Ethan,

    What was your reason for switching from Qiagen minElute purification to AMPure XP beads in those steps?

  2. ethanomics permalink

    Probably the biggest reason is we have a 60 ml bottle of AMPure beads. But I would also like to make the protocol as economical as possible and the AMPure beads are less money. I don’t know what is better, I didn’t do a side-by-side comparison here but it worked well. I’ve checked AMPure recovery of low ng quantities of DNA and it was very good. It is fine to use the Qiagen minElute columns at every step except the last one, but my hunch is AMPure is better.

    I may do some library preps this week and I will do an extra one with significantly less enzyme to see if I can cut costs there.

    Ethan

  3. gillt permalink

    I cannot get a straight answer from NEB as to why they use Klenow fragment polymerase as well as T4 polymerase for end repair in expression libraries but not in their library prep kitss for NGS. The NEB kit for expression libraries has the klenow but their blunt end repair mix for library prep for NGS only has T4 polymerase and kinase. Is it the size of the DNA or the mechanical shearing?

  4. ethanomics permalink

    This has actually been something I’ve put some thought into. I use what was recommended in the pre-TruSeq Illumina kits but I am skeptical that this is actually well optimized. NEB says both T4 and Klenow are good at making blunt ends (at different temperatures though) and given given your experience with what NEB is saying/doing I appears they don’t know what works best. I think there is room for improvement when it comes to end repair. The bottom line is I don’t know the answer to your question but I would guess they don’t know at NEB either. Most all protocols are never throughly optimized. People get the to point where it works and that is good enough. For the most part we are biologists and methodology is not what we are interested in. But with ChIP-seq being able to use smaller quantities of DNA would be very advantageous and help us answer biological questions.

  5. Mattia permalink

    Thanks for sharing your experience ethan.
    Am I correct to think that the PEG in the first two ampure purification is to abolish the size selection?

    Thanks

    M

  6. ethanomics permalink

    Hi Mattia, ChIP fragments can be pretty small so I figured it was best to keep the concentration of PEG high. It also helps reduce costs by using less AMPure beads.

    Ethan

  7. Mattia permalink

    That seems reasonable; have you tried the new Diagenode beads that allegedly do not have a size selection and are cheaper than a Qiagen spincolumn?

  8. saurabh agarwal permalink

    I understand the reason for T4 Polymerase/Klenow for end repair. There is one advantage and disadvantage of each enzyme but using T4 kinase and T4 polymerase in a sequential addition and incubating at different temperature is more prudent than using klenow.

    1. Reason why Klenow is used is because it has a lower exonuclease activity. T4 has a very high exonuclease activity and that it is why it should be used at temperature <16 or preferably 12C. But at 12 or 16, T4 Kinase has a minimal activity. So thats why if a single step method should be used, it should be Klenow + T4 Kinase for simultaneous blunting and phosphorylation.

    2. But use of klenow causes a bigger problem due to its strand-displacement activity and will generate single stranded fragments from nicks which is not good as it starts replicating the DNA.

    Thats is why I first add T4 kinase+ Atp (1X T4 DNA ligase Buffer) for 1 hour at 37 followed by cooling the reactions on ice and adding T4 Polymerase (Only 0.3ul or 1unit), dNTPs and NEB Buffer 2 +BSA to the reaction to a final of 1X NEB Buffer 2+ 500mM each of dNTPS. Blunting reactions are then carried out at 16 C for 10 mins and 12 C for 20 mins. The reaction is purified by Minelute to remove any T4 DNA polymerase which will inhibit dA tailing or ligation by its exonculease activity.

  9. Una permalink

    Hi Ethan,

    Thank you for sharing such helpful information.

    I have a question about the adapters you mentioned in the protocol.

    Did you order adapters separately from Illumina? I think they do not offer the oligo only now.

    And I guess you did not make the library by truseq DNA preparation kit, as you mentioned the enzymes are from NEB.

    So I am wondering how you got those oligos…

  10. ethanomics permalink

    I just order them from IDT with standard desalting and then anneal them according to the protocol. No special purification.

  11. Rob Alba permalink

    Hi Ethan,

    If one had to make 100 ChIP-seq libraries using your entire protocol (End Repair through Amplify Library by PCR), what would be the cost per library?

    Rob

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