HTG Single Cell Sequencing

The High-Throughput Genomics Core Facility supports the Fluidigm C1 system, an automated solution for single cell genome and transcriptome analysis. Integrated Fluidics Circuits (IFC) have been designed by Fluidigm to capture up to 96 cells per experiment and enable DNA or mRNA amplification from the individual cells at the capture site. Following harvest of the amplified product, one can perform quantitative PCR or alternatively Illumina sequencing libraries can be prepared to more fully characterize the transcriptome or genome of individual cells.

Integrated Fluidics Circuits

Integrated Fluidics Circuits (IFC) have been designed for the Fluidigm C1 System that enable capture of single cells from targeted size ranges including 5-10 um, 10-17 um and 17-25 um. Circuits are available for amplification of either mRNA or genomic DNA.

Cell Concentration

The researcher should provide the core facility with a suspension of cells in growth media or PBS at a concentration of 100-300 cells per microliter. Each circuit is capable of capturing up to 96 individual cells and following capture, the IFC is scored to define the positions that contain cells and the number of cells within each capture location. Fluidigm suggests that that successful capture of 40-60 cells tends to be a more realistic expectation of capture efficiency.

Filtration of Cell Suspension

Cell suspensions of dissociated tissues may contain cell aggregates in addition to fully dissociated cells. Aggregates that enter an IFC can potentially clog the fluidics passages which reduce the cell capture efficiency and adversely affect the delivery or reagents for cell lysis, cDNA synthesis or amplification of nucleic acids. To reduce the likelihood of fluidics clogs, we recommend filtration of the cell suspension. A filtration unit may be used that allows passage of cells and particles up to 20 um larger than the targeted cell size (i.e. Miltenyi Biotec 30 um Pre-Separation Filter cat#130-041-407).

Scoring of the IFC Following Cell Capture

Following passage of a cell suspension through an integrated fluidics circuit, the IFC is ejected from the C1 instrument. The IFC enables staining and scoring of the 96 capture sites within the IFC by microscopy prior to cell lysis and nucleic acid amplification.

Harvest of Amplified Nucleic Acids

Following nucleic acid amplification, the amplified products are harvested from individual wells in the IFC enabling harvested products to be linked to individual scored cells. The concentration of amplified cDNA or genomic DNA is measured using a Qubit dsDNA HS assay (Invitrogen cat#Q32851). Typical concentrations range from 0.15 to 1 ng/ul.

Library Preparation from C1 Amplified cDNA

Amplified cDNA can be used to construct sequencing libraries using the Illumina Nextera XT Sample Prep kit thus enabling transcriptome analysis of individual cells. Following quantitation of the cDNA yield from individual cells, a member of the core facility will contact the researcher to review the data and define samples that should go forward with library preparation and Illumina sequence analysis.