The C1 preps individual cells for genomic, transcriptional, and quantitative gene expression comparisons. In addition, the C1 system allows for captured cells to be stained and examined by microscopy for viability, surface marker, or reporter expression prior to cell lysis. It offers a significant technical advancement towards our understanding of cellular diversity that was previously unattainable.
The C1 uses a microfluidic circuit to capture individual cells, based on size. Once the cells are captured they can be examined individually using a microscope so that cell viability and/or phenotype can be documented. The capture process uses about 1000 cells in suspension and randomly catches about 10% of these to fill the 96 capture sites on the circuit. There are 3 available circuits, for cells 5-10um, 10-17um, and 17-25um. Once the cell capture is inspected and considered worthy, the circuit is placed back into the C1 for cell lysis and synthesis of cDNA.
For cDNA synthesis, we use the Clontech SMART-Seq v4 Kit for Ultra Low RNA Input, which generates cDNA for each individual cell. Typically, this process is run overnight and the cDNA is harvested the following morning. Once the cDNA is made, it should be checked for quality and quantity. A typical extraction will produce about 10ng or more of cDNA harvested in 13uL. This material can be used for downstream PCR or for making deep-sequencing libraries.
Please contact Victoria Best or Dave Dunaway for planning your single-cell experiment. Flow Core staff will operate the C1 but the investigator must be present to visually inspect the IFC post-capture. Prior to your first experiment, we require documentation of cell size, and cell buoyancy, as these parameters must be taken into consideration during the cell capture.