Protocol for Microfluidic System to Automate the Preparation and Fractionation of the Nucleic Acids in the Cytoplasm Versus Nuclei of Single Cells

Materials and Reagents

1. Microfluidic chip fabrication
   Fabricate a microfluidic device (Figure 1) from polydimethylsiloxane (PDMS) (Dow Corning, Sylgard^® 184 Silicone Elastomer Kit) and glass slides by using soft lithography. The nominal channel width and depth of the microfluidic device are 90 μm and 35 μm, respectively. The basic design is similar to the microchannel used by Shintaku et al. (2014) except for a downstream branch channel. We have made available the CAD file for the device in the supporting information.
   
   
   Figure 1. Schematic of channel geometry. A. The letters (a to f) in the figure identify the various channel sections. Vi (for I = 1 to 5) refer to the voltages applied to platinum electrodes placed in respective reservoirs. The numbers also refer to the reservoirs, e.g., 1 = I, 2 = nNA, 3 = cNA, 4 = B, and 5 = V. B. The lengths of channel sections in the chip.
   
   
2. Reagents for extraction
   Prepare following solutions in UltraPure DNase-/RNase-free deionized (DI) water (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977 ). Prepare buffer solutions under DNA/RNA free and DNase/RNase free environment (e.g., clean room conditions)
   
   1. Cells (A20, mouse B cell lymphoma)
      
   2. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
      
   3. Triton^TM X-100 (Sigma-Aldrich, catalog number: X100 )
      
   4. Tris (Sigma-Aldrich, catalog number: 93362 )
      
   5. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
      
   6. Polyvinylpyrrolidone (PVP) (Sigma-Aldrich, catalog number: 437190 )
      
   7. HEPES (Sigma-Aldrich, catalog number: PHG0001 )
      
   8. Sucrose (Sigma-Aldrich, catalog number: S0389 )
      
   9. Leading electrolyte (LE) (see Recipes)
      
   10. Trailing electrolyte (TE) (see Recipes)
       
   11. Cell suspension buffer (see Recipes)
       
       
3. Reagents for RT-qPCR and qPCR
   The reagents described here are for an experiment similar to Kuriyama et al. (2015) wherein we performed quantitation of gDNA for the nucleus and cytoplasmic RNA for the cytoplasm. However, we note the general lysing, fractionation, and preparation protocol described here is well applicable to other studies (e.g., analysis of nuclear versus cytoplasmic fractions of RNA from single cells).
   1. TaqMan^® RT-PCR mix (2x)*
      
   2. TaqMan^® gene expression assay
      
   3. TaqMan^® RT enzyme mix (40x)*
      
   4. TaqMan^® RNA-to-CT^TM 1-step kit (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4392653 )
      
   5. Primer set (forward primer, reverse primer) (TaqMan^® probe)
      
   6. DI water
      
   7. RT-qPCR master mix for cytoplasmic RNA analysis for two reactions (see Recipes)
      
   8. qPCR master mix for gDNA analysis for one reaction (see Recipes)
      *Note: TaqMan^® RNA-to-CT^TM 1-step kit contains both reagents.

Equipment

1. Microscope
   A phase contrast microscope fitted with a CCD camera enables monitoring the lysing and cytoplasmic nucleic acid fraction focusing via isotachophoresis (ITP) [see on-line movies by Garcia-Schwarz et al. (2012) for a typical on-chip ITP experiment.]. It also helps monitor migration of nucleus and its fractionation from the cytoplasmic fraction in the ITP zone. Avoid using fluorescence-based visualization, e.g., intercalation dye, which may interfere specificity and stringency of downstream assay.
   
2. High voltage sequencer
   A voltage sequencer (e.g., LabSmith, Inc., model: HVS448-3000D) automates the lysis, extraction, and fractionation via ITP.
   Note: A current measurement helps monitor the ITP zone migration and fractionation of the nucleus.
1. Send the voltage sequence to the voltage sequencer.
   
   
   
   
2. Connect the voltage outputs to platinum wire electrodes that will be placed to the reservoirs.
   

Procedure