I’ve entered, all vim and vigor, into a number of pacts with the devil. They often start off with, “what an elegant technique! I could do that!”
The comfortable part of passing a Ph.D. is that you should have learned by then that you know a certain complement of techniques, and that you are capable of assimilating others. If you think by the time you get to a postdoc that you’ve learned all the tools you need, you have another think coming. The contemplation of learning and refining tricks and tools of the trade for the rest of your professional life is something you had better enjoy if you wish to become a scientist.
So I recognized the sentiment that Alan Moore voiced in this interview:
I think the amount of work we contribute to our enjoyment of any piece of art is a huge component of that enjoyment. I think that we like the pieces that engage us, that enter into a kind of dialog with us, whereas with film you sit there in your seat and it washes over you. It tells you everything, and you really don’t need to do a great deal of thinking.
Sometimes, though, I would rather certain protocols less resemble works of literary art and get down to brass tacks already. When I think I spent something like four years getting SAGE to work under our particular technical constraints, I am a little pessimistic about our current blockages. As promising as JoVE is, it does not yet have the same back catalogue as IMDB (although the latter site only has the abstracts, after all).
absolutely. I was attempting to do CHIP right up until leaving the lab—would have done it too, if only I could have found the person who owns the Stratalinker in time…
Hi Heather,
I’m not sure if you’re saying that you’d like a video protocol for ChIP or whether a written step-by-step protocol would be helpful….?
At Nature Protocols, we’re exploring the idea of video protocols (we already have some author-provided videos in the form of Supplementary Information). And we’re always very interested to hear what actual hands-on researchers think makes a useful video protocol!
We have published a few ChiP protocols (though no videos), both on the peer-reviewed (subscription required) and user-generated (free to access but not edited or peer-reviewed) parts of our site. Just go to our homepage and enter ‘ChIP’ in the search box. The upper box on the search results page provides links to the peer-reviewed content, the lower box provides links to the freely available user-generated content. Hopefully there might be something in there that will be of use to you? If not, you could always try posting a request for help on our Discussion Forum here on Nature Network?
We are also always happy to listen to suggestions for additions to the peer-reviewed content of our site, so if you think there’s something obvious we should be covering please do let us know!
Good luck with learning the new technique!
I think your man is watching the wrong films.
Thanks, Dorothy… I think what I was saying in a very roundabout way was that I derive a certain amount of enjoyment about adapting a complex technique to my individual constraints. But sometimes I wish things were easier and less need to customize.
I think ChIP doesn’t need a video, much like any protocol entailing microscopic chemical reactions and transfer of liquids from one place to another. But we see with the recent proliferation of protocols and kits which are the steps that are user-dependent and which can not be tinkered with.
Lee – aside from perhaps The Life of Others, which films have you seen lately that have made you imagine anything off screen, eg. really think?
I haven’t yet seen The Lives of Others; but this recently took me sideways for one. Actually, reading the two paragraphs either side of the one from which you lifted the quote, I’m largely with him, although not so negative on film as an art form as he seemingly comes across.
As for filmed protocols… they would presumably be depicted as working first time. Not very imaginative, as they seldom do. (Err, do they…?)
No, quite right, they don’t. I really do love films (generally, not always specifically). And am sorry I missed your excellent and provocative post the first time around, but glad you linked to it again. On the other hand, were you thinking about all these things during the movie, or afterward? Where I differ from Alan Moore’s take is that much as I also enjoy comics, I wouldn’t put them on a mental workout level with a novel or even a non-fiction book, no matter how provocative. There is enjoyment, and perhaps more goes in from the reader than for a movie, but as our visual sense is quite so dominant (at least, mine is) it still overwhelms my imagination, for the most part.
Hi. Before, during, after. Some bits anyway. For me, the first test of a good film is if I come out and want to see it again. Because there was more going on than you might have first absorbed; or it sets you off on a train of thought; and/or something else triggers or connects (such as what I considered a misleading news piece; was the post ‘provocative?’).
You’re right – the book is more ‘work’. (Films = lazy reading?)
I am a bit late, but thanks for the link to LSTOTT… things are a bit stagnant over there so it’s appreciated. :)
ChIP-seq is, well… the “seq” part of it is hardly routine, but we’ve done a bunch of them now. The ChIP part, however, is way outside our scope – we get clients to do that nasty dirty work and send us the resulting vanishingly small amounts of clean and shiny DNA.
But – I could put you in touch with some local friends and experts if it might help.
Thanks, Richard! But we’re not having any problems much with using eg. a histone trimethyl K4 antibody – more with the antibodies against your run-of-the-mill homeobox-containing transcription factors that we actually want to study. Anyone who does ChIP will tell you that the black box encloses the time of fixation and the “quality” of the antibody. Meaning you just have to try to optimize by trial and error. That’s where we’re at.
For one of them we sent the DNA for sequencing and the results are kind of – eh. So I am trying to massage them into shape by changing the call criteria and so forth (only 1/3 of the sequences were mapped by ELAND, for example, though by hand most do).
Mmm. I just spoke with our bioinformatics gurus and they described our results using ELAND as, and I quote, “horrible”. For ChIP-seq they’ve had much more luck using Bowtie. Most other people I’ve spoken with use MAQ, and BFAST looks like it will be good, but we’ve had some problems getting them to compile and run happily (on SUSE Linux on our cluster, although MAQ seems ok on a desktop running Ubuntu – very strange, since I’m told the kernel is the same).
This is all with Illumina reads BTW. For Roche/454, our gurus still think Bowtie would be a good choice. You’re using Roche as I recall… maybe?
Hey – good example of NN being useful. The folks we subcontracted to used MAQ as well, with similar results to ELAND, and I was going to try the alignment module of CisGenome, but with some trepidation in my heart. The Bowtie algorithm looks good, in particular this:
“Bowtie allows the user to select the number of mismatches permitted (default: two) in the high-quality end of a read (default: the first 28 bases) as well as the maximum acceptable quality distance of the overall alignment (default: 70).”
That way I could in theory conduct a certain number of simulations.
I probably should hook up with a real bioinformaticist; I keep saying that from time to time. We’ve got a Ph.D. student on board (at least part time) for another aspect of the project, dealing with enhancer motifs, but I think I need a guru.
I really appreciate your speaking to someone about it! (We decided against Roche even if it is locally available, because for the mapping in ChIP-Seq, the number of reads [5 million versus 400K] in theory gives us better resolution for generating peaks. Even only a third of the reads.) If we had the possibility of generating large quantities of the ChIP’d DNA, I’d probably be happy with the Roche and its less ambiguous sequence attribution, but as it is, it seems difficult to drag our signal out of the background noise.
Mm. Our Roche instrument spat out nearly 1.2 million reads last time we ran it – I imagine others are seeing similar performance with the latest set of upgrades and such. But you’re right, more is better and if you can get the error tolerances right, you shouldn’t need long reads for ChIP-seq. Or so I’m told. ;)
I’m still trying to find someone who remembers our first Roche read mapping project – I rather suspect it was done with batch-BLAST though, or perhaps 454 did the mapping themselves. It was before my time here, or early enough in my tenure that I didn’t know about it.