We are using C1 Single-Cell auto prep system(from Fluidigm) to capture, lyse, cDNA synthesis and pre-amplify mature full length mRNA or DNA. Our future goal is to develop assays to apply all available genomics assays at a single-cell level.
1. C1 (C1 Single -Cell Auto Prep System)
The C1™ System is a benchtop automated single-cell isolation and preparation system(lysis and pre-amplification) for genomic analysis. The C1 System provides an easy and highly reproducible workflow to process 96 single cells for DNA or RNA analysis. This new system enables you to rapidly explore unique attributes of individual cells without the technical variability of manual single-cell workflows.
Using an integrated fluidic circuit (IFC) chip, researchers can lysis, reverse transcribe and amplify RNA. Three different IFC chips enable this material to be analyzed on either a Fluidigm Biomark using real time PCR technology or an Illumina HiSeq using ultra high throughput RNA-Seq technology.
1a. IFC Chip (C1™ Single-Cell Auto Prep Integrated Fluidic Circuit)
*It is recommended that measure your using cells to increase the capturing efficiency (or to avoid getting no or multiple cells in a nest).
|Cell diameter*||Possible cell types*|
|Small||5-10 µM||stem cells and WBC|
|Medium||10-17 µM||iPS, progenitor cells, neurons, and others|
|Large||17-25 µM||fibroblasts, keratinocytes, cardiomyocytes, smooth muscle cells, and others|
Flow of C1 System
1b. Harvest Pre-Amplified cDNA and Size Range Check
Pre-amplified cDNA is harvested from C1 chip using a multi-channel pipette & transfer to a 96 well PCR- plate. The size distribution is checked by DNA High sensitivity kit (Bioanalyzer, Agilent), and followed by concentration check with a Picogreen® assay kit.
– Bioanlyzer eletropherogram of pre-amplified cDNA (using SMATer kit off C1 chip)
1c. Library Prep Using Nextera XT Kit
We are using (& recommended by Fluidigm) Nextera XT DNA Sample Preparation Kit, enabling sequencing-ready libraries to be generated in a short period of time. In short, DNA is simultaneously fragmented and tagged with sequencing adapters in a single step, using standard lab equipment. Tagmented 96 samples can be differently indexed with a Nextera Index kit & PCR amplified. The amplified indexed libraries are pooled, bead normalized and cleaned. The prepared libraries are ready for next step (Bioanalyzer QC and qPCR). Libraries prepared with a Nextera kit are compatible with all Illumina sequencers
– Bioanlyzer eletropherogram of tagmented, indexed, pooled & bead normalized RNA-seq libraries on DNA-HS kit (using NEXTERA XT)
2. Other systems
We are looking into different systems (Drop-Seq & ICELL8 Single-Cell), we can develop customized protocols with, since C1 is not an open system to run a customized protocol. Please contact the Lab Manager if you have any question.
We have a protocol to try out on manually isolated individual cell in 0.2mL PCR tube. Please contact the Lab Manager if you want to try us out.