Single Cell RNA Sequencing Library Preparation

Single cell RNA sequencing has become an important tool for measuring cell to cell transcript variability on a transcriptome scale. Chosen by Nature as the 2013 ‘Method of the Year’, single cell RNA sequencing allows one to measure unique cellular processes as well as the effect of local environments or stimuli in transcript expression.

Most RNA-Seq studies are performed on a population level, where the transcriptomes of millions of cells are averaged. In certain sample types, such as stem cells, circulating tumor cells (CTCs) and other rare populations, sufficient material cannot be obtained for analysis on such a scale. Unique preparation methods are required to make libraries from picograms of RNA. Techniques to prepare single cell RNA libraries typically rely on 1) homopolymer tailing, 2) template switching, 3) in vitro transcription, 4) rolling circle amplification.

Genohub has a lot of experience helping design low input or single cell RNA-sequencing experiments. Contact us to take advantage of our complementary consultation service. You can also search for and identify providers who offer single cell RNA-Seq services.

See Genohub's up-to-date list of available Single cell RNA-seq library prep services

Single Cell RNA-Seq Kits

Clontech SMARTer Ultra Low Input RNA Kit

The SMARTer kit from Clontech uses a template switching approach called ‘Switching Mechanism at 5’ End of RNA Template’ to generate full length cDNA libraries from as few as 1-1,000 cell or 10 pg – 10 ng of total RNA. The single tube protocol works on whole cells and serves as a module upstream of Illumina and Ion compatible library preparation protocols.

Protocol Overview:

  1. Total RNA or 1 – 1,000 cells
  2. First strand synthesis leaves an extended tail on the 5’end of the cDNA
  3. Template switch by the addition of a 5’primer that anneals to extended cDNA region in step 2
  4. Extension by reverse transcriptase
  5. PCR amplification