Home-made ABi SOLiD Total RNA-Seq ??

[dcyounge]

This kit is too expensive. Anyone with knowledge, information, or a friend who might know a guy who once made a home-made version of this kit, please let me know ! Thanks!

[pmiguel]

This kit is too expensive. Anyone with knowledge, information, or a friend who might know a guy who once made a home-made version of this kit, please let me know ! Thanks!

Well, you just need to buy some T4-RNAligase2 and whomp up a batch of adapters. Check out the patent disclosure for details of the chemistry.

Tricky though. I wouldn't touch it unless I had 100+ libraries to make.

Do you need strand specificity? You could just by the Illumina RNA TruSeq kit and substitute SOLiD adapters for the Illumina adapters. Much cheaper! They even throw in the poly A+ RNA purification.

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Phillip

[ETHANol]

Agree, just buy the Illumina kit and substitute in your own adapters. The cost of synthesizing the adapters is minimal. We are getting good data with using 1/3 of all the reagents.

On second thought, it's not that simple since the SOLiD adapters are not Y-shaped. I don't really know much about SOLiD library preparation.

[pmiguel]

Agree, just buy the Illumina kit and substitute in your own adapters. The cost of synthesizing the adapters is minimal. We are getting good data with using 1/3 of all the reagents.

Austerity measures?

On second thought, it's not that simple since the SOLiD adapters are not Y-shaped. I don't really know much about SOLiD library preparation.

It doesn't matter that much. You have to make 4 oligos/adapter pair instead of 2. I would base them on the new SOLiD frag library adapters since they have T-overhangs. Then during enrichment PCR the A-A and B-B homo-adapter molecules snap-back to form intra-strand loops with the adapters forming the stem. This makes them much less available to primers and they basically don't amplify vs. the A-B and B-A molecules. It is just a very simple form of suppression/pan-handle PCR at work.

Okay, that does mean you have to use SOLiD primers instead of PPC from the Illumina kit to do your enrichment PCR. Still the outlay for oligos will not be too bad.

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Phillip

[dcyounge]

Thanks folks ! Found an epicentre kit that will be better for our experiments. But trying to figure out what their secret "degradase" is .. any thoughts ? see Figure 1 http://www.epibio.com/posters/Enhanc...ome_ABRF12.pdf

[pmiguel]

Thanks folks ! Found an epicentre kit that will be better for our experiments. But trying to figure out what their secret "degradase" is .. any thoughts ? see Figure 1 http://www.epibio.com/posters/Enhanc...ome_ABRF12.pdf

The "TAP" degradase is "Tabbaco Acid Pyrophosphatase", of course. The other degradase -- well there are the usual suspects: RNAseIII, RNAseH (although you would have to anneal the RNA to some DNA first), and someone mentioned in one of these forums using limited digests of S1 nuclease or Mung Bean Nuclease to give the desired 5' -phosphate, 3' -hydroxyl. Looks like at least one company (other than Epicentre) went that way.

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Phillip