Illumina RNA-Seq Preparations

[clostridium40]

I'm just getting into RNA-Seq, and had a question about the library preparations for Illumina HiSeq. I know that Illumina sells a kit that will take the isolated mRNA samples and convert them to dsDNA during library preparation. However, I was wondering if anyone knows or has experience with using other kits (ie. Invitrogen) to convert the mRNA into dsDNA then do the library preparation for the Illumina. Any thoughts or advice on the subject would be very much appreciated.

[pmiguel]

Unless you need the strand information, go for the TruSeq RNA kit. It starts from total RNA (0.1-4 ug) and produces indexed libraries for about $70/sample in kit reagents. You need to buy some other reagents (eg Ampure). But we spend more than that just on ribo-depletion for other methods.

I thought maybe the ribo-depletion would not work that well. But first set of libraries we sequenced, it looks like rRNA reads are at most 2%.

There is an "A" and a "B" kit -- each with 6 adapters. This is really the only complaint I have with the kit:
(1) For the "A" kit, one of the adapters, "AD03", I think, is the same as the multiplex phiX adapter. So, if you want to spike in 1% phiX into each lane instead of wasting 1/8th of your run on phiX, you have to skip samples with that index.
(2) Why not just offer a 48 index kit? They have all the indexes for the small RNA kit. Irritating!

--
Phillip

[clostridium40]

I'm just getting into RNA-Seq, and had a question about the library preparations for Illumina HiSeq. I know that Illumina sells a kit that will take the isolated mRNA samples and convert them to dsDNA during library preparation. However, I was wondering if anyone knows or has experience with using other kits (ie. Invitrogen) to convert the mRNA into dsDNA then do the library preparation for the Illumina. Any thoughts or advice on the subject would be very much appreciated.

Phillip,

Thanks for the valuable information. I have one other question, the Illumina kit does ribo-depletion based on poly-A tails on the mRNA right? So, I'm working with prokaryotic mRNA, so do you think that this kit is still worth it, since I will still have to run through several other ribo-depletion kits prior to the Illumina kit. Since we are not Illumina customers, I haven't been able to find a price for the Illumina TruSeq RNA kit do you know how much it costs? Thanks again for all your great information.

[tboothby]

Phillip,
I have one other question, the Illumina kit does ribo-depletion based on poly-A tails on the mRNA right? So, I'm working with prokaryotic mRNA, so do you think that this kit is still worth it, since I will still have to run through several other ribo-depletion kits prior to the Illumina kit. Since we are not Illumina customers, I haven't been able to find a price for the Illumina TruSeq RNA kit do you know how much it costs? Thanks again for all your great information.

Hi,
Yea the TruSeq kit first has you isolate total RNA and then do a polyA purification step.

However, I have used this kit without their RNA isolation reagents. So I supplied pre-purified RNA and just started at the cDNA/fragmentation step.

If your only reason for not wanting to use the TruSeq kit is that you have to do a different RNA isolation protocol, then I would still just buy the kit (especially if this is you first time trying RNAseq)

Cheers,
T

[pmiguel]

Phillip,

Thanks for the valuable information. I have one other question, the Illumina kit does ribo-depletion based on poly-A tails on the mRNA right? So, I'm working with prokaryotic mRNA, so do you think that this kit is still worth it, since I will still have to run through several other ribo-depletion kits prior to the Illumina kit. Since we are not Illumina customers, I haven't been able to find a price for the Illumina TruSeq RNA kit do you know how much it costs? Thanks again for all your great information.

A kit is about $3350. It can make 48 libraries with 6 different indexes.

The price is still pretty good, even if you don't use its ribo-depletion steps. But, yeah, you might prefer to look other places since you don't want polyA isolation in your case.

--
Phillip