S1 File The Protocols for preparing the laboratory practical lessons

A549 cell culture and transfection with pCMV3-Spike-GFPSpark plasmid

Thaw bacteria ( E.coli JM109 strain) on ice for about ten minutes (use about 100 µl aliquots);

Resuspend the pellet in 5 ml of buffer P1 added with 100 µg/ml RNase A and Lyse Blue solution

Add 4 ml of buffer P2, shake by inversion for 4 times, until the solution became blue or light blue, incubate 5 min at RT

Add 5 ml of buffer P3, shake by inversion 4 times (comment: white precipitated particles will form) incubate on ice for 15 min

Centrifuge for 30 min at 4°C, 3,000 rpm; take the supernatant (containing the plasmid DNA)

Equilibrate a QIAGEN-tip adding 4 ml of QBT buffer, allow column to become empty by gravity flow, then add the supernatant and allow it to enter the resin by gravity flow

Wash the column 2 times with 10 ml of QC Buffer, allowing the buffer to move through the QIAGEN-tip by gravity flow

Elute the plasmid with 5 ml of QF buffer into a clean tube

Elute the plasmid with 5 ml of QF buffer into a clean tube

Centrifuge 1.5 ml tubes at 12,000 rpm for 30 min at 4°C, discard the supernatant by inverting 1.5 ml tubes and dry tubes using a lab paper

Add 400 µl of 75% EtOH to each tube and centrifuge at 12,000 rpm 4°C for 10 minutes

Mix bacteria by finger vortexing and add 10 ng of pCMV-C-GFPSpark by slowly pipetting (no more than twice);

Remove the ethanol and allow the pellets to dry in the air, under the hood

Resuspend the plasmid DNA pellet in TE (Tris-EDTA pH = 8)

Incubate on ice for 30 min and then at 42°C for 1 min

Put on ice for 2 min

Add 200 µl of LB (Luria-Bertani)-medium (Triptone; Yeast Extract; NaCl) without antibiotic

Incubate at 37°C for 1 hour, keeping in slow shaking (about 90 rpm);

Incubate at 37°C for 1 hour, keeping in slow shaking (about 90 rpm);

The day after pick a colony and grow bacteria carrying the pCMV-GFP plasmid in 100 ml of LB-medium (Luria Bertani medium) with antibiotic (Kanamycin, final concentration 25 µg/ml) at 37°C under shaking until the desired density is reached

Take about 50 ml of culture medium containing bacteria grown up to the plateau (turbid medium); centrifuge at 3,000 rpm for 30 min at 4°C; remove the supernatant by inverting the 50 ml tube

A549 cell culture and transfection with pCMV3-Spike-GFPSpark plasmid

Detach A549 cells, when are at confluence, from a starting T25 flask. Briefly: remove cell supernatant, wash the cells once with 5 ml of DPBS, add 1 ml of trypsin-EDTA and incubate at 37°C for 5 min. Add 1 ml of FBS to inactivate trypsin action and add 8 ml of DMEM to restore the starting volume

Remove the supernatant, wash the well with 1 ml of DPBS, add 200 µl of trypsin and incubate at 37°C for 5 min, add the same volume (200 µl) of FBS, restore the starting well-volume with 600 µl of DMEM

Centrifuge detached cells at 1,200 rpm for 8 min RT

Centrifuge detached cells at 1,200 rpm for 8 min RT

Centrifuge again as indicated at point “k”

Remove the supernatant and resuspend the pellet by adding 300 µl of DPBS

Remove the supernatant and resuspend the pellet by adding 300 µl of DPBS

Plate A549 cells in a 12-well plate at 35-40% confluence one day before the transfection using DMEM + FBS 10% as medium

Incubate cells for 24 hours at 37°C, 5% CO₂

The day after, remove the old medium, and replace it with 1 ml of new medium composed by DMEM + FBS 10%. The procedure should be performed at least 30 min before the transfection. At the day of the transfection cells must be at 70-80% of confluence;

Prepare a mixture containing 1 µg of pCMV3-Spike-GFPSpark plasmid, 1 µl of PLUS Reagent and 47.5 µl of opti-MEM. Incubate the mixture at RT for 5 min

Add 2.5 µl of Lipofectamine LTX to the mixture and incubate at room temperature for 30 min

Add 2.5 µl of Lipofectamine LTX to the mixture and incubate at room temperature for 30 min

Stir the plate and incubate at 37°C, 5% CO₂for 24 hours

The day after observe cells at fluorescence microscope using FITC filter (Excitation 490, Emission 525);

RNA extraction.

Detach cells adding 200 µl of trypsin to each well, incubate for 5 min at 37°C, add the same volume of FBS to inactivate trypsin and restore the starting well volume with 600 µl of DMEM

Take the upper aqueous phase without touching or drawing the interface or the pink organic phase, and transfer it into a new 1.5 ml RNase-free tube: work under a chemical hood

Add 500 µl of 100% RNase-free isopropanol to precipitate the RNA and mix well by inverting the 1.5 ml tube. (Comment: Isopropanol is very volatile and can drip, to avoid this, prime the tip before taking the isopropanol);

Incubate the samples at RT for 10 min

Centrifuge at 12,000 rpm, for 15 min, at 4°C. A pellet containing the RNA will be obtained

Discard the supernatant with the pipette

Add 900 µl of 75% RNase-free ethanol, pre-cooled on ice, (Comment: the ethanol is very volatile and can drip, to avoid this, prime the tip before taking the solution)

Gently invert the 1.5 ml tubes to wash the pellets. Observe carefully the size of the pellet obtained, this is essential to establish the volume of water to be used to resuspend the pellet

Centrifuge at 12,000 rpm, for 5 min, at 4°C

Discard the supernatant with the pipette

Allow the RNA pellets to dry in the air under the hood for about 10 min

Collect cells and centrifuge at 1,200 rpm for 8 min at RT

Resuspend the RNA in 20 µl or more of RNase-free H₂O, pipetting several times (Comment: the volume of water to be added may vary based on the size of the pellet and will be established after careful observation of the pellet)

Store on ice, if analyzed immediately, or freeze at -80°C

Remove supernatant and wash the cellular pellet with DPBS

Lyse cellular pellets adding 900 μl of TRI Reagent under a chemical hood: immediately after addition, pipette quickly to make the system homogeneous (comment: no white fragments of the pellet should be present)

Incubate the samples for 5 min at room temperature (RT), keeping them in the rack

Add 200 µl of RNase-free chloroform, under a chemical hood. (Comment: chloroform is very volatile and tends to drip; to avoid this, prime the tip before taking the chloroform);

Mix vigorously by inversion for 15 sec, until the solution becomes homogeneous and the two phases can no longer be distinguished

Incubate the samples at RT for 2-3 min, keeping them in the rack

Centrifuge at 12,000 rpm, for 15 min, at 4 ° C. The mixture separates into 3 phases:

-Upper aqueous phase: transparent, contains RNA

-Whitish semi-solid interface: contains DNA and proteins

-Lower organic phase: pink, contains DNA, proteins and lipids