If you are planning to use this system for single cell sequencing applications it is important to understand the landscape of technologies and approaches available to researchers. Some approaches may be better suited depending on the researchers budget, resources, desired data or outcome, expertise and the biological samples or systems under consideration as well as the environment in which experiments might need to be carried out. Before embarking on building this project I suggest a full reading of the following papers which represent a snapshot of popular massively parallel single cell transcriptional profiling techniques:
1) Macosko et al. "Highly
Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets" 2015, Cell 161 1202-1214
2) Klein et al. "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells" 2015, Cell 161 1187-1201
3) Zheng et al. "Massively parallel digital transcriptional profiling of single cells" 2016, Nature Comm.
4) Gierahn et al. "Seq-Well: portable RNA sequencing of single cells at high throughput". 2017, Nature Methods
The instrument described is composed of 3D printed parts affixed with electronic and pneumatic components. The instrument replaces the following components of a typical [Drop-seq] setup:
2) Syringe pumps (3 are required)
3) Magnetic stirrer
4) Microscope camera
The Instrument costs about $550-600 to build this is about 1/2 to 1/4th the cost of single syringe pump which is typically used to generate flow in research-level microfluidic setups.
The instrument uses a Raspberry Pi camera coupled with a laser diode lens to serve as the microscope. The video feed of the experiment is broadcast through a custom written GUI running on a Raspberry Pi 2 model B connected to a touchscreen. Pressure is generated via a micro air pump connected to pressure regulators and PCB mounted micro solenoid valves. The pump and solenoids are controlled through the GUI program which interface through the PCB to the Raspberry Pi. Pressure is monitored through two PCB mounted pressure sensors and pressure values are displayed to the screen.
Fluid is contained within three cryovials (1.8mL (2) and 5.0mL (1) ) which house the 2 aqueous flows (cells and microparticles) and the fluorinated oil respectively. The vials are sealed with custom caps which interface with the output of the solenoid valves (micro air pump) and internal tubing that dips into the fluid within the vial and connecting to the microfluidic chip.
Microparticles are stirred via actuation of a stepper motor (controlled via EasyDriver) that has been affixed with a permanent rare-earth magnet situated on the base of the instrument beneath the fluid vials. When a magnetic stir disc is placed within the microparticle vial the stir disc rotates due to local inversion of the magnetic field via stepper motor shaft rotation.
A typical [Drop-seq] experiment takes about 35-45 minutes for 1mL of each aqueous component. The result is the (Poisson limited) encapsulation of single cells and barcoded microparticles for single-cell RNA-seq. After which the standard [Drop-seq] protocol can be followed to generate single cell libraries.
In addition to the hardware components of the instrument, to perform a [Drop-seq] experiment you will also need:
- Microfludic chip (design mDS_v12.dwg)
- Barcoded microparticles ("Drop-seq beads" Chemgenes Corp. Wilmington, MA)
- Micro tubing PE2 (Scientific Commodities)
- Razor blade or exacto knife
- Nunc Cryovials 1.8mL & 4.5mL (Thermo Fisher Scientific)
- Magnetic stir disc (V&P Scientific, Inc.)
- Tweezers for tubing placement
- 50 mL conical for collecting droplets
- BioRad QX200 fluorinated oil
- Flexible USB reading light (aids in visualization of the droplets)
- Associated reagents, equipment, pipettes, and tips for downstream molecular biology reactions and sequencing
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