TITLE: S2 File  The Protocols of the laboratory practical lessons
AUTHORS: Jessica Gasparello, Chiara Papi, Matteo Zurlo, Lucia Carmela Cosenza, Giulia Breveglieri, Cristina Zuccato, Roberto Gambari, Alessia Finotti

[DESCRIPTION]
The protocols here described are related to the sections presented in Figure 1B focusing on the practical activity performed by the students. All the steps are presented and critically commented when deemed useful. The reagents used during the preparation of these protocols are indicated as just an example, as all the reagents can be purchased from any company.

[STEPS]
SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
1. In a graduated cylinder prepare 1 L of   solution diluting    stock solution; with deionized water: 20 mL  of TAE 50x and 980 mL  of deionized water are required

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
2. Prepare the tray to add agarose solution, adding two   well combs

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
3. Weight 0.8 g  of    powder and add it into a glass flask

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
4. Add to the glass flask 100 mL of   

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
5. Boil the suspension in a microwave for same minutes, until the solution will be completely clear
SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
6. Add 10 µL  of FluoroVue 10000x nucleic acid gel stain and stir the solution until it will be completely homogeneous (agarose solution will become light yellow)

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
7. Add slowly the agarose solution to the gel tray and carefully remove possible bubbles
SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
8. Let the agarose solution solidify for 30 min  at room temperature

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
9. In the meantime, prepare the unknown sample adding a standard volume of sample: 2.5 µL  (Comment: this volume corresponds to about 700 ng  of plasmidic DNA or RNA), 2 µL of tartrazine tracking dye and 7.5 µL of nuclease free water. Mix the sample by pipetting three or four times

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
10. Spin the sample to disrupt possible bubbles and keep the sample on ice until the loading
SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
11. When the gel is solidified, setup the electrophoresis apparatus: remove the tray and set the gel into the electrophoresis chamber, add TAE 1x solution to the electrophoresis chamber, carefully remove 1 mm-well combs
SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
12. Load 10 µL  of unknow sample to the gel

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
13. Run the agarose gel with TAE 1x buffer, at 80V for 40 min 

SECTION: Part 1: Quality control of (i) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (ii) RNA extracted from A549 and Spike-plasmid transfected A549 cells
14. Check the results at UV-transilluminator 

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
15. In a graduated cylinder prepare 1 L  of TAE 1x solution diluting TAE 50x stock solution with deionized water: 20 mL of TAE 50x and 980 mL  of deionized water are required

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
16. Prepare the tray to add agarose solution, adding two   well combs

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
17. Weight 1.8 g  of agarose powder and add it into a glass flask

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
18. Add to the glass flask 100 mL  of TAE 1x

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
19. Boil the suspension in a microwave for same minutes, until the solution will be completely clear
SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
20. Add 10 µL  of FluoroVue 10000x nucleic acid gel stain and stir the solution until it will be completely homogeneous (agarose solution will become light yellow)

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
21. Add slowly the agarose solution to the gel tray and carefully remove possible bubbles
SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
22. Let the agarose solution solidify for 30 min  at room temperature; in the meantime, prepare the sample adding 2 µL  of Orange loading dye to 10 µL  of PCR product

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
23. Spin the sample to disrupt possible bubbles and keep the sample on ice until the loading
SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
24. When the gel is solidified, setup the electrophoresis apparatus: remove the tray and set the gel into the electrophoresis chamber, add TAE 1x solution to the electrophoresis chamber, carefully remove   -well combs

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
25. Load 10 µL of sample to the gel or 55 µL  of Gene Ruler 50 bp

SECTION: Part 2: RT-qPCR analysis of (a) plasmid DNA, (b) RNA from A549 cells and (c) RNA from A549-Spike cells
26. Check the gel at UV-transilluminator
SECTION: Reverse transcription
27. Thaw the unknown samples and spin to put the sample at the bottom of the 0.2 mL  tube. Keep the sample on ice for all the experience

SECTION: Reverse transcription
28. Dilute the sample with 10 µL  of nuclease free water

SECTION: Reverse transcription
29. Add 4 µL  5x Prime Script Buffer and the same volume of Random 6 mers 100 micromolar (µM) 

SECTION: Reverse transcription
30. Add 1 µL of Oligo dT primer 50 micromolar (µM) 

SECTION: Reverse transcription
31. Add 1 µL  of Prime Script RT Enzyme Mix 1. The final volume of 20 µL  will be reached. (Comment: enzyme must be stored in a refrigerated block, to avoid repeated freeze and thaw cycles)

SECTION: Reverse transcription
32. Pipette up and down three or four times with a p20 pipette set at 20 µL 

SECTION: Reverse transcription
33. Spin down the sample with a microcentrifuge
SECTION: Reverse transcription
34. Incubate the sample within a thermal cycler set at 37°C for 15 min  for complementary DNA synthesis, then incubate at 85 °C  for 5 min  to denature the reverse transcriptase enzyme. After the thermic program the sample must keep on ice. (Note this is a stop point, if required sample can be store at -20 °C  for several days). 
(Comment: during the thermic program of the reverse transcription, the preparation of PCR sample will be started). 




SECTION: RT-PCR
35. Add at the bottom of 0.2 ml tube  16 µL of nuclease free water

SECTION: RT-PCR
36. Add 1.5 µL  of each primers concentrated 50 ng/µl

SECTION: RT-PCR
37. Carefully add 1 µL  of DNA generated at point 1 and keep on ice

SECTION: RT-PCR
38. Add 1 µL  of Wonder Taq Polymerase. The final volume of 30 µL will be reached. (Note for organizers and students: enzyme must be stored in a refrigerated block, to avoid repeated freeze and thaw cycles)

SECTION: RT-PCR
39. Pipette up and down three or four times with a p200 pipette set at 30 µL 3

SECTION: RT-PCR
40. Spin down the sample with a microcentrifuge
SECTION: RT-PCR
41. Incubate the sample within a thermal cycler setting the following amplification program:
-95 °C 3 min initial denaturation and polymerase activation)
-95 °C 15 s denaturation phase)
-60 °C30 s  annealing)
-72 °C 30 s elongation)
-Steps 2, 3 and 4 were repeated for 25 cycles






SECTION: RT-PCR
42. At the end a final elongation phase of 72 °C  for 5 min min was added. After the thermic program the sample must keep on ice. (Comment: this is a stop point, if required sample can be store at -20°C for several days)

SECTION: RT-PCR
43. 10 µl of obtained PCR products, will be loaded on the 1.8 Mass / % volume agarose gel

SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
44. Thaw the sample and pipette up and down two or three times to mix the sample
SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
45. Add 1 µL of sample diluent (in our case nuclease free water) onto the optical sensor and click ‘read blank’

SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
46. Clear the optical sensor with deionized water
SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
47. Withdraw 1 µL  of the sample and add directly onto the optical sensor

SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
48. Click ‘read sample’ and note the two absorbance values: A260 and A280
SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
49. After 0.8 Mass / % volume  agarose gel visualization calculate sample concentration considering that: 
plasmidic DNA: 1OD of absorbance at 260 nm correspond to 50 µg/ml; RNA: 1OD of absorbance at 260 nm correspond to 40 µg/ml
SECTION: Part 3: Quantitative control of (a) the pCMV3-Spike-GFPSpark plasmid (Spike plasmid) and (b) RNA extracted from A549 and Spike-plasmid transfected A549 cells
50. Using the value of absorbance at 280 nm calculate A260/A280ratio to determine purity of the sample