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  • advanT
    replied
    Hi ETHANol, pmiguel, I'm glad you're both on this thread because I have questions for both.

    I tried your chip-seq protocol (thanks for making this available by the way) with 10 ng of fragmented genomic DNA (not chip) and was able to get libraries at 220 bp however I see a higher molecular weight band between 400 and 500 bp on my agarose gel. These are the only two bands that I see on the gel. After my gel size selection I only expected to see the 220 bp band. The higher MW band is twice as intense as my expected band. Any suggestions?
    pmiguel, I've read some of your posts about these mysterious higher MW bands and am wondering if this is the same thing.

    Are there any magic bullets to eliminate these? Has anyone tried sequencing their sample, leaving it in there? Unfortunately because they are so close you can't really separate one from the other unless you do a gel cut. I'm just worried about that because I considered myself fortunate to even see the low input on a gel.

    Leave a comment:


  • jlove
    replied
    [QUOTE=jlove;49657]
    Originally posted by ETHANol View Post
    0.25 μM means 0.25 μM for each adapter primer. You might consider diluting them a little more say to 0.125 μM. I'm not sure what the concentration of the adapters form Illumina are..

    Regarding adapter - I made my own TruSeq adapters and I wanted to get them the same concentration as Illumina's...so I ran them out at a few concentrations on the smRNA chip (boiled first) alongside the Illumina ones. For what it's worth, my 8-15 uM (each) dilutions looked most similar to the Illumina ones. I had also made 25 uM, 20 uM and the peaks were much higher

    Leave a comment:


  • jlove
    replied
    duplicate post, sorry
    Last edited by jlove; 08-22-2011, 06:14 AM. Reason: duplicate post

    Leave a comment:


  • pmiguel
    replied
    Originally posted by ETHANol View Post

    [...]

    Phillip, I think you may be on to something. But since the goal is not to make long PCR fragments but short ones in the neighborhood of 300-500 bp, it doesn't sound exactly right. But the overhang sounds like a reasonable explanation. Maybe the p5 and p7 binding sites are repeated and that enhances attachment to the flow cell and cluster generation. Or maybe the extended length increases yield with the Agencort beads. I don't know but I am curious. There has to be someone that can mass spec them. Such a simple thing to do.
    I don't have a mass spec handy. (By which I mean I would have to submit it to another core on campus.) I heard that by doing a limited Snake Venom Diesterase digestion + mass spec you can determine oligo sequences.

    Seems like cloning some PCR "enriched" TruSeq amplicons and Sanger sequencing would be one way to go. But that would be getting a little "meta" -- kind of an Easter Egg hunt, courtesy of Illumina, Inc. Maybe someone has already done this, to QC a library? If so, maybe they could slake our curiosity?

    --
    Phillip

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  • xp8
    replied
    In a test I have to sequencing the DNA fragments as short as possible (above 40 bases).

    There is a question:
    What is the bottom line of size that we can sequence with TreSeq system?
    Particularly, If I use 75bp X 2 sequencing, can I do sequencing as short as 40 bases (inset size)? or I have to sequence >75 bp insert?

    Your opinion will be very well appreciated.

    Victor

    Leave a comment:


  • ETHANol
    replied
    Originally posted by jlove View Post
    Great protocol! Question about adapter concentration. Is 0.25 uM stock the concentration of the combined adapters or each one? Is it basically the Illumina tube of adapter at 25 uM diluted 1:100?

    Thanks!
    0.25 μM means 0.25 μM for each adapter primer. You might consider diluting them a little more say to 0.125 μM. I'm not sure what the concentration of the adapters form Illumina are.

    Phillip, I think you may be on to something. But since the goal is not to make long PCR fragments but short ones in the neighborhood of 300-500 bp, it doesn't sound exactly right. But the overhang sounds like a reasonable explanation. Maybe the p5 and p7 binding sites are repeated and that enhances attachment to the flow cell and cluster generation. Or maybe the extended length increases yield with the Agencort beads. I don't know but I am curious. There has to be someone that can mass spec them. Such a simple thing to do.

    Leave a comment:


  • pmiguel
    replied
    I was thinking about this again. How about if the PPC oligos have ~60 nt 5' "heads" on them. For example 60 A's, then the actual priming site oligo. The result, after a couple of cycles, would be each product amplicon as a single stranded molecule would have a poly A tail on one end and a poly T tail on the other. (Those are just examples, could be other sequences.)

    What for? This would allow a stem structure to form. The stem would be less available for primer annealing, once it formed. Because the formation of this stem would be unimolecular, it might be faster than the bimolecular primer annealing step. The stability of the stem loop structure would, in part, be determined by how large the loop (the insert) was. This is the basis of the the "PCR suppression effect" or sometimes called "pan-handle PCR". It can be used to compensate for PCR's short product bias.

    --
    Phillip

    Originally posted by pmiguel View Post
    Here is some evidence that the TruSeq PCR primers are just the entire TruSeq adapter oligos:
    We ran an Agilent RNA pico chip on 0.2 ul from the "PPC" (PCR primer cocktail) from:

    A DNA TruSeq A kit:


    An RNA TruSeq Bkit:


    Here is the ladder electropherogram for that chip:


    The sizes are a little longer than expected (58 and 63 nt for the A strand and B strand oligos, respectively). But I guess that is down to mediocre accuracy for the pico chip in that size range.
    --
    Phillip

    Leave a comment:


  • jlove
    replied
    Great protocol! Question about adapter concentration. Is 0.25 uM stock the concentration of the combined adapters or each one? Is it basically the Illumina tube of adapter at 25 uM diluted 1:100?

    Thanks!

    Leave a comment:


  • ETHANol
    replied
    Just to follow up and say the protocol worked great. I've posted the details on my new blog at:
    I got the data back from the ChIP-seq samples I sent out using my TruSeq library preparation protocol. I can definitely say the library preparation protocol works great, clearly better than what Il…

    Leave a comment:


  • pmiguel
    replied
    Probably would work better with p5 and p7, or something similar, like the primers ETHANol designed. Still one can't help being curious as to what the Illumina guys are doing...

    --
    Phillip

    Leave a comment:


  • ETHANol
    replied
    It seemed to work with the primers I designed which are a couple nucleotides longer then the p5 and p7 primers as far as generating a nice smear on the gel. But I'll have to wait until my samples are sequenced to be sure. I'm not sure how long the queue is. I've searched extensively and haven't seen anything.

    Leave a comment:


  • advanT
    replied
    Has there been any problems with just using the p5 and p7 primers? It seems it would be easier and make more sense in terms of Tm compared to these longer primers.

    Leave a comment:


  • pmiguel
    replied
    To be honest, I am mystified as well.

    Tm for the Universal Adapter is 75 oC. About the same for the Index adapter oligo. 60 oC would be a full 15 oC below the Tm.

    --
    Phillip

    Leave a comment:


  • ETHANol
    replied
    All I can say is (some) DNA oligos must run not true to the markers on the RNA chips. It just goes against everything I know about PCR. Sometimes you have to believe the data, but sometimes the data is misleading. Someone needs to mass spec them. We have a mass spec but not the PPC oligos. Weird stuff!!!!!

    And if the oligos are so long, why does Illumina say to use a 60˚ C anneal temp for the PCR? Is there something that I am missing completely?

    Leave a comment:


  • pmiguel
    replied
    I am pretty sure there is no 25 nt oligo in the PPC. 25 nt is the size of the spike in size standard for all lanes of the RNA pico chip. We also loaded the same samples at 5x the concentration. The ~80 nt peak is much larger, but the 25 nt peak is approximately the same area.

    I'll try to remember to run PPC next time a small RNA Agilent chip is run.

    BTW, the chip reports the peak as somewhere between 5-6 ng/ul for a 5x dilution. So roughly 25-30 ng/ul.

    I would hypothesize that the peak is a mixture of the TruSeq Universal Adapter (58 nt) and 6 TruSeq Adapter Index oligos (63 nt). Average molecular weight around 18 ng/pmol. Less than 2 pmol/ul? Seems low. 5 ul are used in the 30 ul PCR reaction. That would be about 20 pmol in total oligos.

    --
    Phillip

    Leave a comment:

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