Library Prep from Limited RNA Samples

[Xeriandros]

I am new to the field of Library Preparation and RNA-Seq in general, and have some questions about the best options for performing transcriptome analyses on my samples.

I have limited amounts RNA (50-80ng total per sample, at 2-4ng/uL) prepared from my samples of interest, and I am looking to prepare Libraries for sequencing with the NextSeq 500 sequencer.

As far as I can tell, my RNA quantities are too low for traditional library preparation, leaving me with a choice between RNA amplification kits or alternative library preparation kits such as the TruSeq RNA Access Kit (which isolates RNA coding regions for sequencing). Is there a clear advantage to choosing one of these strategies over the other? Are there alternative strategies/kits that I am not aware of that might work bettter? I would greatly appreciate any help that you can provide.

[fanli]

There are a number of low input RNA kits that can work with your input amounts. See:

http://www.illumina.com/applications/sequencing/rna/ultra-low_rna_input.html

[Simone78]

if you want to do it cheap and without kits you can use our Smart-seq2 protocol. It works only for mRNA and gives a non-stranded library. See PMID: 24056875; 24385147. It´s optimized for single cells but works also with 1-10 ng tot RNA.

If you have enough RNA (and money!), want a stranded library and all the RNAs (also the non-polyA+) you should use the SMARTer Stranded Total RNA Sample Prep Kit (Clontech).

If you want to do only tag-seq (seq only the 5´or the 3´of the transcripts) you should use either the CEL-seq (PMID: 22939981;24531970) or STRT (PMID: 24363023; 21543516)methods.