Clontech Smart Seq v4 Ultra Low RNA Input

[sajoshi]

Have you used this kit? if you have used it what kind of RNA samples did you use. I have doing some initial experimentation and wondering which RNA would be suitable: RNA from paraffin (FFPE extracted) or RNA from cerebrospinal fluid. Someone is donating me these samples.

Thanks, please advise

[kwalton]

I do this protocol with sorted cells and routinely use only 1ng of total RNA (according to the bioanalyzer pico chip- yes I know it's not quantitative but it's the only measure of concentration I can get as the HS RNA qubit is often too low for my samples) however have used much less and it works well. We also used 10ng of total RNA and found we have better data (less rRNA) with 1ng than with 10ng. What protocol will you go into for library prep? I have not used it for FFPE and the protocol states that it should be used with good quality (RIN>8) total RNA or cells that haven't been fixed. I'm happy to share my experience with you if you need more information. I'm running these samples through the proton using a Kapa kit downstream of Clontech.

[sajoshi]

I will probably use the Illumina protocol because we have Illumina HiSeq.

[jwfoley]

Virtually all the protocols are compatible with the Illumina HiSeq, but if you take a look at how the Clontech kit works, you'll see why it's not compatible with degraded RNA.

[NextGenSeq]

We found that with 1 ng of total RNA we routinely see adapter artifacts.

[sajoshi]

What type of RNA would work best. I have seen sometimes CSF RNA samples have degraded RNA. I work in a core and asking other labs to donate samples for trying out the Clontech kit.

[jteeee2]

Any mostly intact total RNA sample will work with this kit. We have used RNA samples with RIN's as low as 5-6 with success. This kit utilizes the poly-A tail for initial priming, so you are only going to be looking at coding regions of the transcriptome. Input amounts as low as 100 pg have worked in our hands.

If you are using highly degraded material, you could try one of the other low input kits suitable for FFPE style RNA inputs. An example of this would be the Clontech SMARTer Stranded Total RNA Seq Kit-Pico. I have not used this kit so I cannot comment on its performance.

[InDel]

We used Kapa kits for degraded RNA (RIN 2 or 3) and it works.