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Old 04-15-2014, 09:52 AM   #1
peterwang
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Default commercial oligo set for RNaseH depletion of human rRNA?

We are interested to try depletion of rRNA from human samples by the RNase H method of Morlan (recommended in the survey of methods by Adiconis):

Morlan JD, Qu K, Sinicropi DV. Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One. 2012;7(8):e42882. doi: 10.1371/journal.pone.0042882.

Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby MA, Berlin AM, Sivachenko A, Thompson DA, Wysoker A, Fennell T, Gnirke A, Pochet N, Regev A, Levin JZ. Comparative analysis of RNA sequencing methods for degraded or
low-input samples. Nat Methods. 2013 Jul;10(7):623-9. doi: 10.1038/nmeth.2483.

I'm wondering if there is a source of the pooled oligos against rRNA and mitochondrial RNA that is required for this method? Of course we can have them custom-synthesized, but would be nice to try it out first.

Anyone have experience with this method that they want to share?

Thanks,
- Peter
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Old 06-21-2014, 09:22 PM   #2
konglongjidan
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Hi Peter,

We are interested in looking into this method as well. But we haven't found any available commercial ones. So we are thinking of making it ourselves from IDT, but it is a big investment and a bit "risky". Let me know if you have any good thoughts!!!
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Old 06-22-2014, 10:40 AM   #3
peterwang
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Hi konglongjidan:

We did custom-order the oligo set from Adiaconis and tried out the RNaseH depletion method (made in-house, as crude unpurified; note that several oligos contain inosine in place of guanine to break up runs of Gs). It seemed to work very well, as well or better as RiboZero done on the RNA.

If you want to try it out, I could send you some of our oligo mixture (send me a private message).

cheers,
- Peter
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Old 05-31-2015, 11:54 PM   #4
peterwang
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This is just an update in case others come across this thread in a search.
The homebrew RNase H depletion method (using the protocol and oligos from the Adiconis paper) has worked well for us.

However, for those that don't want to spring for the large initial cost of the oligo set, note that the method has been commercialized by New England Biolabs (I have no affiliation) as the NEBNext rRNA Depletion Kit:
https://www.neb.com/products/e6310-n...uman-mouse-rat

I note in passing that the NEB kit uses E coli RNase H rather than the more expensive thermostable RNase H used in the Morlan and Adiconis papers, so it is likely that a homebrew version could also use the cheaper enzyme, but we have not tried this.

cheers,
- Peter
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Old 11-18-2015, 04:06 AM   #5
jbergenstrahle
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Hey,

Is the NEBNext rRNA Depletion Kit still the only commercially available kit that uses RNase H? The experience from our lab is that this method is superior to the capture-and-separate-methods that do not use an enzymatic step.

My NEBNext kit was called back due to "quality issues" and there might be "several weeks" before the problem is fixed. Looking for alternatives.

Quote:
Originally Posted by peterwang View Post
...
Eventually I might try a "homebrew" method like you guys. Peter, I would definitely be interested in a list of the oligos you used!

Cheers,
JB
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Old 11-18-2015, 02:24 PM   #6
peterwang
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to jbergenstrahle:

The oligos we used are from the Adiconis paper in Nature Methods, in an Excel file as Supplementary Table 6:
http://www.nature.com/nmeth/journal/...h.2483-S4.xlsx

There are a couple other companies besides New England Biolabs that now provide RNaseH-based ribosomal depletion (again, I have no affiliation, and have not used any of the products). There may be others that I don't know about.

RiboGone from Takara/Clontech:
http://www.clontech.com/US/Products/...al_RNA_Removal

RiboErase from KAPA Biosystems, but this is only available as part of a library construction kit:
https://www.kapabiosystems.com/produ...seq-riboerase/

cheers,
- Peter
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Old 11-18-2015, 02:40 PM   #7
peterwang
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Still some life in this old thread!
I got the following as a PM, thought I would post reply to the board.

Quote:
I'm also thinking about synthesizing my own oligo set. So my question is that what's your experience with crude oligo you mentioned in your earlier post. Do you mean just desalting? Do you have a rough idea of the residue rRNA % in the libraries with this method in your hand? Any tip on working with this method? Thanks a lot!
Yes, we had oligos synthesized just with desalting, no other purification, and that worked fine.

After depletion, in our RNA-Seq runs we generally saw 1-2% (or less) reads mapping to ribosomal RNA.

I pretty much followed the protocol in the Adiconis paper, though doing only one clean-up using silica column rather than two with SPRI beads. I attach my protocol to this post.

As a side note: It was pointed out to me by our in-house oligo synthesis facility that the oligo sequences we got from the Nature Methods paper by Adiconis contain some inosines (which adds to the cost) in place of guanines. I believe the reason for this is to break up long runs of Gs, which are prone to form G-quartet structures.

cheers,
- Peter
Attached Files
File Type: pdf RNase H Depletion of ribosomal RNA, v1.2.pdf (159.3 KB, 104 views)
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Old 11-18-2015, 11:12 PM   #8
jbergenstrahle
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Thanks Peter for the excellent reply! I will look into these

Cheers,
JB
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Old 11-19-2015, 01:35 AM   #9
kobeho24
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excellent! thanks for sharing such impressive stuff. BTW, any preference of kit shown above? neb, kapa, clontech which one has the best performance?

cheers,
gary
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Old 11-23-2015, 07:15 PM   #10
acdan
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It is so great to see your experience! I am also repeating Adiaconis's protocol. But a minor question raised that is do you find any difference between fragmentating the total RNA before and after rRNA depletion? In Adiaconis's protocol he used fragmentated "low quality" samples.
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