RNA Detection

1 is labeled with a CF640R (Biotium) reporter dye at the 5^0 -

end and has the sequence

50 - mCmUmUmCmGmUmCmCmAmCmAmAmAmCmA

mCmAmAmCmUmCmCmUmGmAmAmGmGmAmCmGm

GmCmAmGmCmGmUmGmCmAmGmCmUmCmUmU-3^0.

The underlined sequences are self-complementary to drive the

formation of the hairpin structure, the sequence in bold is

complementary to the target, while the sequence in italics

hybridizes to Oligo 2. Oligo 2 is labeled at the 5^0 -end with an

Alexa Fluor 750®reference dye and at the 3^0 -end with an Iowa

Black RQ-Sp quencher and has the sequence: 5^0 -

mGmAmGmCmUmGmCmAmCmGmCmUmGmCmCmG-

mUmC-3.

2. Phosphate buffer: 48 mM K 2 HPO 4 , 4.5 mM KH 2 PO 4 , and
   14 mM NaH 2 PO4,pH 7.2.


3. Prep grade gel filtration column (e.g., GE Healthcare Superdex
   200 or Superdex 75).


4. 10,000 MW cutoff centrifugal device (e.g., Millipore Microcon
   YM-10).


5. UV-Vis spectrophotometer.

2.4 Microporation 1. Microporation system (e.g., Thermo Fisher Neon transfection
system).

2. MEM without antibiotics, supplemented with 10% FBS and
   1 GlutaMAX (Thermo Fisher).


3. Resuspension buffer R (Thermo Fisher).


4. Electroporation buffer (Thermo Fisher).


5. Electroporation Gold Tips (10μL size) (Thermo Fisher).


6. Electroporation tube (Thermo Fisher).


7. Microporator Pipette (Thermo Fisher).


8. 8-well chambered cover glass (e.g., Nalgene Nunc Lab-Tek).

2.5 Single-Molecule
Fluorescence In Situ
Hybridization

1. Nuclease-free water.


2. 4 w/v % paraformaldehyde diluted in 1PBS.


3. 70 v/v % ethanol, prepared from anhydrous ethanol.


4. 2SSC.


5. Wash buffer (2SSC, 10 v/v % formamide).


6. Hybridization buffer (10 w/v % dextran sulfate, 2SSC, 10 v/
   v % formamide).


7. d2EGFP FISH probes [7], a set of singly labeled probes that
   are complementary to different regions of the d2EGFP coding
   sequence.


8. Parafilm.

234 Yantao Yang et al.