RNA Detection

7. Transfer 1–1.5 mL of the cell suspension to a microcentrifuge
   tube.


8. Pellet the cells by centrifugation at 200gfor 5 min at 4C.


9. Aspirate the media and gently resuspend the cell pellet in 1 mL
   of 1PBS.


10. Count cells.


11. Pellet the appropriate number of cells for microporation at
    400 gfor 5 min at 4C. 30,000 HT1080 cells are used for
    each microporation (seeNote 6).


12. Aspirate the PBS and resuspend the cell pellet in resuspension
    buffer R at 30,000 cells per 10μL.


13. Add 1μL of RBMBs per 10μL of cells such that the final
    concentration of RBMB is between 0 and 5μM and pipette
    gently to mix (seeNotes 7and 8 ).


14. Microporate 10 μL of the cell suspension (approximately
    30,000 cells) with 2 pulses for 25 ms at 950 V (seeNote 9).


15. Following microporation, transfer microporated cells to a micro-
    centrifuge tube prefilled with 1.5 mL of cell culture media.


16. Pellet the cells at 400gfor 5 min at 4C.


17. Aspirate the media, being careful not to disturb the cell pellet.
    Add 1.5 mL of cell culture media to resuspend the pellet.


18. Repeatsteps 16– 17 another two times (seeNotes 10and 11 ).


19. After the last wash, resuspend the cells in 250μL of cell culture
    media.


20. Seed the cells in one well of an 8-well chambered cover glass
    until the samples are ready for imaging.

3.3 Single-Molecule
Fluorescence In Situ
Hybridization

1. Carefully pipette out the media from each seeded well of the 8-
   well chambered cover glass.


2. Gently wash the cells thrice with 250μL1PBS.


3. After carefully pipetting out the PBS, in each well gently add
   250 μL of 4% PFA prewarmed at 37C.


4. Incubate the cells in 4% PFA for 10 min at room temperature.


5. After removing PFA, add 300μL1PBS to each well. Incu-
   bate the sample in 1PBS for about 5 min. Carefully pipette
   out the 1PBS and repeat this step once more.


6. After removing 1PBS, add 400μL of 70% ethanol.


7. Close the lid and wrap the chambered cover glass with Parafilm
   to minimize evaporation.


8. Store the chamber, protected from light, at 4C for at least
   16 h.


9. Carefully pipette out the 70% ethanol.

236 Yantao Yang et al.