SMarter Low RNA Input Kit

[vanillasky]

So I have used the SMarter Low RNA Input Kit and after illumina sequencing with an NEB kit we found out that the sequencing pretty much failed. Although I've contacted the company regarding the results I have yet to receive any tech support. The following attachment shows the dscDNA output as measured with a High Sensitivity Kit. The pattern resembles their control outcome described in the manual. Any input would help me figure out where things went wrong.

[Simone78]

Why don´t you try Smart-seq2+Nextera instead? It would be much cheaper...at least up to the pre-amplification step! Your RNA seems to be degraded, do you have a positive control ? You could do RNA extraction from bulk, take 1 ng and do 12-14 cycles PCR. You should get plenty of cDNA.

[vanillasky]

I picked this kit because I am working with low amounts of degraded RNA and this kit was specifically developed for low input pg-ng and for samples that are degraded. The RNA was extracted from a marine sediment. What do you mean by 12-14 cycles of PCR?

[Simone78]

I mean that after RT you would need 12-14 cycles of PCR to get enough DNA that you can continue with library prep (and check on the Bioanalyzer).

[efrainrib]

I've been using the SMARTER total RNA-seq kit to make libraries from nuclear RNA, ranging from 1-10ng. Honestly when I go under 1ng my libraries are not there. However, from 1-10 ng we have made over 100 libraries that look like the traces provided at the end of the clontech manual. We've probably tried upwards of 150... so you can do the math and see the throughput. We've sequenced it already and are working on the analysis of the data, and from our end everything about the kit looks as advertised. would be happy to answer any more questions in depth if youre still doing this stuff

[NextGenSeq]

We use the NuGen RNA-Seq V2. If your RNA is degraded use the FFPE RNA-Seq V2.

It works with anything.