Single Cell RNA SEQ

SmartSeq2 RNA-sequencing 

Assays currently offered by the facility include:

Smartseq2 assays for RNAseq of single cells and very small samples. Link to Nature reference.

The workflow for Smartseq2

Sort cells into lysis buffer in PCR plates or tubes, vortex spin and freeze on dry ice. Samples can be stored in our dedicated -80C freezer for upto 3 month before processing.

Defrost samples add reverse transcriptase reaction buffer and produce cDNA. Then add PCR primers and Kapa HiFi Polymerase to amplify cDNA. Purify cDNA and produce Illumina compatible sequencing libraries using Nextera XT technology.

A more detailed worklflow is available here

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Example cDNA prep from a single cell using smartseq2.

Clontech’s SMARTer Ultra Low RNA input Kit

These Clonetech kits allow RNAseq to be performed on small samples and single cells, using a commercial version of the Smartseq2 kits and are compatible with either PCR plates and tubes or the Fluidigm C1 system. SOP

C1 Autoprep Based Single Cell Capture 

Fluidigm C1 Arrays that allow capture of 96 single cells with integrated PCR for amplification for downstream RNAseq or targeted gene expression analysis. Link to Single Cell auto prep system 

The C1

 

schematic