Next generation flow cytometry for next generation genomics
Recent advances in genomics, particularly in next generation sequencing (NGS), deliver in-depth and detailed genomic information at an unprecedented pace. With the BD FACSseq™ cell sorter, today’s scientists can be at the forefront of scientific research by studying heterogeneity in cell samples at the single cell level. The successful, rapid isolation of single cells for genomic analysis is becoming, in many applications, the essential prerequisite for obtaining meaningful results.
Convenient sample loading
Cell suspensions are loaded on the BD FACSseq cell sorter from 12 x 75-mm polypropylene tubes (maximum volume of 4.5 mL). The recommended minimum sample volume is 0.2 mL. The dead volume of the sample line is approximately 26 μL, and automatic sort monitoring enables unattended sorting. Sample agitation is also available.
The new BD FACSseq cell sorter is based on powerful cell isolation technology, refined for more than 30 years by BD. This new kind of instrument exploits the power of flow cytometry, while dramatically simplifying operation so that genomics researchers can easily engage in single cell genomic analysis.
BD designed the BD FACSseq cell sorter with the needs of the genomics researcher in mind:
- Easy identification and isolation of single cells, concentrating on the performance attributes of cell size, live/dead discrimination, cell cycle state, and cell surface markers.
- Effortless ability to achieve the highest yields and purity attainable, as rapidly as possible, to keep cells viable.
Replaceable fluidics path
The fluidics path, including the nozzle assembly and sample line, can be exchanged. A sensor detects when the sample tube is empty and stops sample flow, preventing air bubbles from reaching the nozzle assembly.
Options for single cell selection in genomic studies
At the heart of the system is the flow cell, where cells are maintained in a steady stream of sheath fluid (a Dulbecco’s phosphate buffered saline isotonic solution) that is controlled by the instrument fluidics system. The sample cells are funneled into a single stream so that they pass, singly, through a laser beam, and may also emit fluorescent light based on cell surface or intracellular fluorescent staining. The signals are collected and detected to determine if the cell is of interest and should be sorted.
The sorting process is rapid and can be performed at up to 7,000 cells per second. The BD FACSseq cell sorter can collect the cells into tubes, as well as 96- or 384-well PCR plates. Options for the selection criteria to choose and sort the cell of interest are available from the simple touchscreen software. The instrument is capable of measuring cell size in the range of 1 to 20 μm, and up to three of the cell attributes, for a total of four parameters to select cells.
| Cell Application | Detection Method | BD Kits and Reagents |
| Single cells | Light scatter | Parameter automatically captured by instrument |
| Nuclear stain, cell cycle | Hoechst 34580,
DAPI | Cat. No. 565877,
Cat. No. 564907 |
| Cell viability | Propidium iodide (PI) | Cat. No. 556463 |
| Phenotype markers | Fluorescent cell labelling | BD Horizon Brilliant™ Violet 421, BD Horizon Brilliant™ Violet 650, BD Horizon Brilliant™ Violet 786 |
| Reporter gene expression | TagBFP | Not applicable |
A complete workflow for gene expression in single cell analysis
The combination of the BD FACSseq™ cell sorter and BD™ Precise assays offers a streamlined workflow that can take the preparation of single cells all the way through to next generation sequencing for gene expression studies. BD offers the first targeted RNA-seq platform capable of absolute and direct molecular counting of transcripts.
Starting with the BD FACSseq cell sorter, researchers can begin the gene expression workflow with single cells. Adding BD Precise assays moves single cells through a streamlined methodology that will deliver original mRNA transcript counts for the genes of choice at the single cell level.
Custom robust primer/assay design provides reliable and reproducible performance to result in an all-in-one amplification and library prep optimized for sequencing on the leading NGS platforms. The four simple steps are:
- Isolation of single cells with the BD FACSseq cell sorter directly into a BD™ Precise plate
- A single assay for reverse transcriptase through analysis, with an easy transfer from the BD Precise plate to pooling the samples into a single tube
- Single tube processing of up to 384 samples that are labeled and tracked to finish
- Complete library preparation in a fraction of the time, with no special equipment and using basic molecular biology techniques, to get samples NGS ready
With this streamlined workflow, researchers can easily amp up the lab’s productivity while ensuring that high-quality single cell samples are NGS ready, for obtaining a new level of accuracy for gene expression data.
Gene expression in single cells
Isolation of single cells from a mixed cell population using the BD FACSseq™ cell sorter, followed by gene expression analysis using BD™ Precise assays
The left cytogram (Panel A) shows the flow cytometry analysis of mixed cancer cells. Jurkat cells, a T leukemia cell line, and T47D cell line, a breast cancer cell line, were mixed at a ratio of 80%:20%, respectively. The mixed cells were stained with EpCAM BV421 and CD3 BV650. Only Jurkat cells stained positive with CD3, whereas only T47D cells stained positive with EpCAM (Panel B). The mixed cells were randomly sorted into each well of a 96-well plate with one single cell per well. Each single cell was lysed in each well, and the mRNA content was barcoded with two types of indices, one that uniquely labels each single cell and another molecular index that labels each transcript during the reverse transcription step.
Panels A and B
Panel C
We interrogated approximately 100 genes that were carefully selected to distinguish each cell population. After pooling the products from two 96-well BD Precise assay encoding plates, one sequencing library was generated and run on an Illumina® MiSeq instrument. A streamlined computational pipeline was created, and automated data analysis was performed on the Seven Bridges Genomics Cloud Platform. Principal component analysis separated the cells into two clusters (Panel C). The genes that were highly expressed in Cluster 1 and Cluster 2 corresponded well with Jurkat (Cluster 1) and T47D (Cluster 2) cells(Panel D). These results demonstrate simple and unambiguous deconvolution of cellular types using a high-throughput single cell genomic sequencing approach, and BD Precise assays combined with the powerful single cell sorting capabilities of the BD FACSseq cell sorter.
Panel D
Technical innovations enable easy single cell isolation with minimal setup time
The total internal reflection (TIR) innovation has produced a self-aligning sort head that is compact, low cost, and easily removable for cleaning or replacing in multi-user environments. Unique to the BD FACSseq™ cell sorter, the TIR technology is an innovation driven by busy genomic labs that require instruments to be at the ready with minimal setup time.
On traditional sorters, the light emitted from a cell is measured perpendicularly to the incident light. Optimal alignment of the laser and the detectors to the stream can require manual alignment of both the laser and the stream. However, for the BD FACSseq cell sorter, we have developed a novel method (TIR) to collect the emitted light trapped inside the stream (see schematic right). The light is reflected upward to be captured by the collection optics. Collecting light trapped in the stream as opposed to light that passes through the stream has four major advantages:
- The sort head is self-aligning
- The flow cell allows for easy accessibility for replacement or cleaning, especially in multiuser environments where sample carryover might be of concern
- The instrument has high light collection efficiency on a par with conventional cytometers
- The technology eliminates the need for expensive optics
Optical jet stabilization means true walk-away single cell collection
The BD FACSseq cell sorter automatically detects perturbations in the stream, stabilizing the point in the stream where droplets containing a single cell of interest break off. Droplet formation is set, monitored, and adjusted continuously, allowing for a completely autonomous turnkey, self-stabilizing sorter that reliably deposits single cells into each well of a PCR plate.
Review and track the sort data for individual cells
The index sorting feature of the BD FACSseq cell sorter correlates the individual cell with its flow cytometric phenotyping so that researchers can return to review the sort data for each cell that is contained in the collection vessel (for example, a 96-well PCR plate). An FCS (flow cytometry standard) software file is available that contains all the sort deposition and the tray position information on an event-by-event basis. This innovative feature allows gene expression data from BD™ Precise assays to be precisely tracked back to the flow characteristics of the specific cell.
The flow cell is removable for cleaning, when sample carryover might be of concern, especially in multi-user environments.
A software file stores all the sort deposition and the tray position information on an event-by-event basis.
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