library prep with low input mRNA: sonication conditions?

[CSO]

Hello all,
I am planning to do Illumina RNA-seq from flow-sorted cells. This means starting the prep with as little as 5 - 10 ng of mRNA.

If possible, I'd like to avoid using the Illumina TruSeq kit and use what we have on hand. This means fragmenting with a bioruptor. Does anyone have suggestions for the sonication settings?

The basic protocol I am following is designed for 10 - 20x more input than I will have. I know the fragmentation conditions scale with input, but I don't know how and I want to be careful with the material I have reserved for testing.


Thanks in advance!
CSO

[NextGenSeq]

Use the Covaris since it does not have a lower concentration limit.

The specs for the Bioruptor are a minimum DNA concentration of 1 ng/ul

[CSO]

Thanks! I will try to find a Covaris. I had not considered minimum concentration.

Per my original question: I assume fragmenting with the Covaris is also subject to concentration, where lower concentrations will require less sonication?

[nucacidhunter]

Per my original question: I assume fragmenting with the Covaris is also subject to concentration, where lower concentrations will require less sonication?

Covaris guide has given shearing settings for 100ng-5ug input in one tube. Occasionally I have used down to 1ng gDNA with no adverse affect on library.

[CSO]

Covaris guide has given shearing settings for 100ng-5ug input in one tube. Occasionally I have used down to 1ng gDNA with no adverse affect on library.

Thanks! So it looks like input amount matters less for Covaris, but quality as determined by size does matter (i.e. gDNA is going to be much longer than dsDNA generated from mRNA).

For reference, here's the protocol for the Covaris S220/E220 Focused-ultrasonicator. The last page contains inoformation about DNA input
<http://covarisinc.com/wp-content/uploads/pn_400103.pdf>


Alternatively, I may have to fragment RNA prior to cDNA synthesis. Here's the Covaris protocol for mRNA or total RNA fragmentation:
<http://covarisinc.com/wp-content/uploads/pn_400086.pdf>

[NextGenSeq]

It makes no sense to me to shear RNA. We generate full length cDNA and then shear the cDNA.

[CSO]

Would you mind telling me your settings for the Covaris, as well as your typical cDNA concentration? Thanks!