Single-cell Gene Expression Profiling of Mouse Stem Cells With Fluidigm Biomark^TM Dynamic Array

Figure 1. Workflow for Single-Cell Gene Expression Profiling in Mouse Stem Cells using Fluidigm Biomark^TM Dynamic  Array. The main basic steps of this protocol are: Single cell sorting on 96 well plates, specific target amplifycation, chip loading, single cell gene expression RT-PCR and data collection. This Fluidigm Dynamic Array integrated fluidic circuit chip (IFCs) contains a network of microfluidic channels, chambers and valves that automatically assemble individual PCR reactions. More specific information about this particular chip can be obtained from http://www.fluidigm.com/single-cell-gene-expression.html.

Materials and Reagents

1. Mouse ES cells (E14 Cell Line) (ATCC^®, catalog number: SCRC-1040^TM )
2. Low-EDTA (0.1 mM EDTA) TE buffer (Teknova, catalog number: T0227 )
3. CellsDirect^TM One-Step qRT-PCR kit (Life Technologies, Invitrogen^TM, catalog number: 11753-100 )
4. Taqman Gene Expression Assays of the genes of interest for your study (Applied Biosystems)
   Note: TaqMan^® Assays are probe and primer sets based on 5’ nuclease chemistry using TaqMan^® MGB (minor groove binder) probes.Search your Taqman gene expression assays here: https://bioinfo.appliedbiosystems.com/genome-database/gene-expression.html
   1. Select the type of experiment you are conducting. Click In gene expression
   2. Select the type of assay you want. click in all gene expression assays
   3. Select what specie you want to target. Click on mouse since our samples are Mouse ES cells.
   4. Enter the target information. Add the gene symbol or entrez gene symbol or entrez gene  ID of the gene you want to analyze its expression.
      
5. 96.96 Dynamic Array Chip for Gene Expression (Fluidigm, catalog number: BMK-M-96.96 )
6. Gene expression 96.96 Dynamic Array sample & loading reagent kit (Fluidigm, catalog number: 85000802 ), which contains:
   1. Two tubes of 2x assay loading reagent (Fluidigm, catalog number: 85000736 )
   2. Two tubes of 20x GE sample loading reagent (Fluidigm, catalog number: 85000746 )
   3. 20 syringes of Control Line Fluid (150 μl)

7. TaqMan^® universal PCR master mix (Applied Biosystems, catalog number: 4304437 )
   
8. Dulbecco's Phosphate-Buffered Saline (DPBS) (Life Technologies, catalog number: 14190-136 )
9. TryPLE express solution (Life Technologies, catalog number: 12605-010 )
10. 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies, catalog number: D1306 )
11. Propidium iodide (PI) (Life Technologies, catalog number: P-3566 )
12. 2x Assay Loading Reagent (Fluidigm, catalog number: 85000736)
13. 2x TaqMan^®Universal PCR Master Mix (Applied Biosystems, catalog number: 4304437)
14. 20x GE Sample Loading Reagent (Fluidigm, catalog number: 85000746 )
    

Equipment

1. 96-well semi-skirted plate, 0.2 ml wells, straight raised sides (USA scientific, catalog number: 1402-9200 )
   
2. Optical adhesive film (Applied Biosystems, catalog number: 4311971 )
   
3. PCR-8-Tube Strips & Strip Camps, natural (USA Scientific, catalog number: 1402-2500 )
   
4. TipOne 0.1–10 μl extended-length filter tips (USA Scientific, catalog number: 1120-3810 )
   
5. TipOne 101–1,000 μl extended-length filter tips (USA Scientific, catalog number: 1122-1830 )
   
6. TipOne 1–200 μl graduated filter tips (USA Scientific, catalog number: 1120-8810 )
   
7. Fluorescence-activated cell sorter (FACS Aria II; BD Biosciences, catalog number: 643756 )
   
8. Multichannel pipettes (10-100 μl Eppendorf Research^®, catalog number: 38-3122000043 ; 0.5-10 μl catalog number: 38-3122000019 )
   
9. Thermal cycler (Veriti^TM 96-well Applied Biosystems, catalog number: PN 4375786 )
   
10. The Integrated Fluidic Circuit IFC-Controllers
11. The MX model primes and loads 48.48 chips
    
12. The HX model primes and loads the 96.96 chips.
    
13. Centrifuge (Eppendorf, catalog number: 5810 )
    

Procedure