RNA-Seq: cDNA prep and fragmentation questions

[ngcrawford]

I'm developing libraries for RNA-seq on the Illumina platform. I have a couple of questions:

1.) After you have made double stranded cDNA from mRNA should you see a bump on a bioanalyzer RNA Pico chip? I see a small bump in the fragmented RNA after I precipitate it, but not when I run a sample of the ds cDNA. I'm assuming that this is because the ds cDNA is substantially more dilute than the fragmented RNA. Is this a 'normal' result?

2.) I'm using Ambion's RNA fragmentation kit to fragment my RNA. Has anyone had success with this kit? If so what were your fragmentation parameters?

3.) At what sort of starting quantities of total RNA have people had success generating libraries for RNA sequencing? I'm using ~ 10 ug of total RNA.

Thanks in advance for your help!

[Melissa]

Don't really understand your first question. Hmmm... why do you run cDNA samples using an Agilent RNA chip?

3rd question, 10ug total RNA is the norm.

[ngcrawford]

Thanks for the suggestions/help Melissa.

I used a RNA pico because that's the most sensitive chip they've got at the core facility I'm using. Supposedly they should have some of the new Agilent High Sensitivity DNA Kits in soon. I'll switch to those when they arrive.

[Melissa]

The Agilent bioanalyzer software should give different results depending on the option you select and chip you use. As a result, mRNA and total RNA cannot be run together on the same chip.

Have you look at the Bioanalyzer gel profile? Is there any smear on the size range? The RNA chip is probably not sensitive enough to detect the smear.

[ngcrawford]

I run the RNA pico chips, really just the same as an RNA nano, but with conditioning solution at the mRNA setting.

I can only see a smear in the resolubilized mRNA pellet (post fragmentation). It's very small. I've attached a copy of the BioAnalyzer output.

My guess is that the mRNA purification isn't working correctly, but it's difficult to troubleshoot when I don't know what successful RNA-seq BioAnalyzer result looks like. Perhaps, someone can post one?

[elaney_k]

Have you gone through the whole Illumina sample prep protocol yet? If you're starting with ~10ug total RNA there's a good chance that you will see a smear on the post-ligation gel purification step which is your fragmented RNA. I've been able to visualise this smear for total RNA inputs of ~ 5ug.

[Melissa]

This is really bad. Seems like your mRNA has degraded.

Refer this file for mRNA profile. It should look like a broad binomial curve. You will want the peak of this curve shifted on the right because it means that you'll get longer mRNA. The smoother the curve, the better the quality.

It's better you convert your electropherogram x axis to bp rather than s. Then you can tell the size range of your smear.

[ngcrawford]

elaney_k and Melissa,

Thanks for your help. It looks like it's my mRNA purification that's the issue. My total RNA actually looks pretty nice. I'll work on optimizing the total to mRNA step.

It's also good to know that I should see a bit of a smear in the post-ligation gel purification step. I haven't observed one yet so clearly something is going wrong before that point.

- Nick

[elaney_k]

It's also good to know that I should see a bit of a smear in the post-ligation gel purification step. I haven't observed one yet so clearly something is going wrong before that point.

- Nick

Hi Nick,

I've uploaded a picture of 2 RNA-seq post-ligation samples that were generated from 5ug total RNA so you have a reference. I will say though that I don't always see this smear but if your input is 10ug Total RNA it's more likely that you will,

hope this helps,

Elaine

[ngcrawford]

elaney_k,

Could you tell me what total RNA purification method you're using?

- Nick

[elaney_k]

Hi Nick,

For cultured cells definitely Qiagens RNeasy kit and for tissue samples I'm pretty sure it was an RNeasy kit post tissue disruption (I can get the specifics if you need it). The subsequent mRNA from both gave similar smears on the gel.

Elaine

[ngcrawford]

Elaine and Melissa,

Thanks for all your help. I believe I have managed to make my first Illumina RNA-seq library. I need to clone and sequence some of the fragments, but the gel of the PCRed size selected adaptor ligated fragments looks good (nice smeary band at ~ 200 bp).

It turns out my difficulty in RNA extraction stemmed from an error in my mRNA purification protocol. The original protocol said to add 15 ul of Dynabeads where I really needed to add 100 ul. I also used fresh, never frozen, total RNA. This modification resulted in ~ 150 ng total mRNA from 10 ug total RNA. Basically, I used the protocol from the Giland Lab with some modifications.

http://giladlab.uchicago.edu/data/RNASeq_v2%202.doc

Whew. And, thanks again.

- Nick

[elaney_k]

Hi Nick,

Congrats on the library!

The Illumina protocol says to use 15ul of Sera-mag Oligo-dT beads which are now supplied in the RNA-seq kits. It's possible that they're better at purifying mRNA than the Dynabeads or supplied at different concentrations and hence the difference in amounts used. I use the Illumina protocol of 15ul of Sera-mag Oligo-dT beads to produce our libraries with no problems.

Elaine

[RSK]

Hi Nick,

I am wondering how you decided on the fragmentation times with the Ambion fragmentation kit? I am starting with 100ng of RNA and am not sure if I should do a titration curve for that small amount since Ambion's protocol is for larger amounts.

Thanks,
RSK

[ngcrawford]

RSK,
I used total RNA and not mRNA for the titration curve. I had ~ 10 ug (= 10,000 ng) to work with so I could just run the fragmented RNA on a gel.

5 min at 70C seemed to work pretty well, but you could do shorter.

- Nick