Single Cell RNA SEQ
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
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