TITLE: Library transfection
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This protocol details the transfection in 6-well plates.

[BEFORE_START]
Be sure that the HEK293T-LLP-iCasp9-blast cell line (Matreyek et al 2020) expresses BFP at a level of at least 98% before beginning a transfection (see Purity Sorting HEK293T LLP iCasp9 Cells). 
VAMP-seq library transfections can be scaled up to 10cm2 culture dishes. Here we detail the protocol for transfection in 6-well plates.
Allow Fugene 6 to come to room temperature before use.

[STEPS]
SECTION: Day -2
1. Two days before the planned transfection, passage HEK293T-LLP-iCasp9-blast culture and seed 2 T-175 flasks (more or less, depending on the number of cells needed).  

 

SECTION: Day 0
2. In the morning (or night before), lift and plate cells at 50% confluence in 4 6-well plates. Let the plates sit in the BSC for 30 min after plating, before carefully placing them in the incubator.
SECTION: Day 0
3. Set up two sets of 1.7-mL microcentrifuge tubes for each sample (2 per well)
SECTION: Day 0
2.1. At least 7-8 hours after plating the cells, transfection can begin. 
 

SECTION: Day 0
2.2. For a 6-well plate, aim to plate 500,000 - 600,000 cells per well. 
SECTION: Day 0
3.1. Label tubes with the sample/control name and A or B for the corresponding tube.
SECTION: Day 0
4. To the set of B tubes, add OptiMEM first and Fugene 6 second as outlined in the table below. 
 
 
 
SECTION: Day 0
4.1. It is very important to add Fugene 6 second in this step.
SECTION: Day 0
4.2. When adding Fugene 6 to the OptiMEM, do NOT touch tube walls with the pipette tip. 
SECTION: Day 0
4.3. Mix by pipetting gently up and down.
SECTION: Day 0
5. Allow the B tubes to incubate at Room temperature for at least 5 min
SECTION: Day 0
6. To the set of A tubes, add OptiMEM first, Bxb1 plasmid second, and sample plasmid last  as outlined in the table below. Mix by pipetting or a short vortex.
 
 
 
SECTION: Day 0
7. Add the DNA mixture from Tube A TO the Fugene mixture in Tube B and mix by pipetting.

 

SECTION: Day 0
8. Allow the transfection mixture (now in Tube B) to incubate at Room temperature for at least 15 min but no more than 30 min.
SECTION: Day 0
9. Remove the plated cells from the incubator and label the wells for each control and the plates for library transfection.
SECTION: Day 0
10. Add the transfection mixture from Tube B to the culture media in a dropwise manner to the appropriate well. Swirl gently to mix.

 

SECTION: Day 0
11. Place the plates in a 5% CO2, 37 °C incubator for 2880 min.
SECTION: Day 2 (48 hours after transfection)
12. Remove plates from the incubator.
SECTION: Day 2 (48 hours after transfection)
13. Lift cells and split the controls 1:5. Combine the library wells by plate into an appropriately sized culture flask.
SECTION: Day 2 (48 hours after transfection)
13.1. This is growth dependent. If the library cells are slow growing, can put 6 wells into one T175 flask (3 flasks total). If the cells are faster-growing, combine 3 wells into one T175 flask (6 flasks total).
SECTION: Day 2 (48 hours after transfection)
13.2. Cultures in T175 flasks should each contain a volume of 30mL D10 media.
SECTION: Day 2 (48 hours after transfection)
14. Add 2 mg/mL doxycycline to each sample and swirl gently to mix.
SECTION: Day 2 (48 hours after transfection)
14.1. Add 2 µL for each control (in a 6-well plate) 

SECTION: Day 2 (48 hours after transfection)
14.2. Add 30 µL to each library T175 flask.
SECTION: Day 2 (48 hours after transfection)
15. Incubate in a 5% CO2, 37 °C incubator for 2880 min.
SECTION: Day 4 (96 hours after transfection)
16. Prepare a portion of all cultures for flow cytometry. Passage the controls if necessary and re-plate. Re-plate the libraries without expanding the culture.
SECTION: Day 4 (96 hours after transfection)
17. On the cytometer, set the laser parameters and gates for live cells and single cells.
SECTION: Day 4 (96 hours after transfection)
18. Create a scatterplot with BFP fluorescence on the x axis and mCherry expression on the y axis.
SECTION: Day 4 (96 hours after transfection)
19. Create a gate for the BFP-negative and mCherry-positive cells (in the top-left corner of the graph). 
SECTION: Day 4 (96 hours after transfection)
20. Collect data for an appropriate number of events (50,000 events) and note the percentage of single cells that are within the BFP-negative, mCherry-positive gate. This is the transfection efficiency.
SECTION: Day 4 (96 hours after transfection)
21. After recording data for all controls and library samples, these cells may be discarded (aspirate into the bleach trap).
SECTION: Day 4 (96 hours after transfection)
22. To the library flasks, add 30 µL of 10 nanomolar (nM) AP1903 (Rimiducid) and swirl gently to mix.

SECTION: Day 4 (96 hours after transfection)
22.1. AP1903 kills cells that express iCasp9. In this case, cells that express iCasp9 were not successfully recombined, and do not contain a variant from the library.
SECTION: Day 4 (96 hours after transfection)
22.2. This step enriches the number of recombined cells by killing those which did not successfully recombine during transfection.
SECTION: Day 4 (96 hours after transfection)
23. Incubate at 5% CO2, 37 °C for 1440 min.
SECTION: Day 5
24. Check the library flasks. If there is a lot of cell debris, aspirate the media from the flask and replace it with 30 mL D10 media and 30 µL doxycycline (2 mg/mL ). 

 

SECTION: Day 5
25. Return the flasks to the incubator.
SECTION: Days 6-8 (or when cells have reached confluence)
26. Continue to check on the library flasks for the next few days. Split the controls as necessary while the library flasks grow.
SECTION: Days 6-8 (or when cells have reached confluence)
27. When library cultures have reached 90-100% confluence, they are ready to sort by fluorescence-activated cell sorting.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
28. Prepare half of the library flasks and half of the control cultures for flow cytometry (see the Purity sorting HEK293T LLP iCasp9 cells protocol for preparation).
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
29. Prepare several (5-8) sets of 4 recovery tubes for the sorted cells. Label each set with numbers 1-4 to represent the bins that the sorter will separate cells into.

Add 2 mL D10 media to each recovery tube.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
30. On the flow cytometer software, open your experiment and create tubes within that experiment for the controls and variant library.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
31. Remove the cap and load the flow tube containing cells into the sample loader. 

Press “Load” on the software. 
Analysis should begin automatically, or press “Acquire data”.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
32. Gate for single cells by gating in the following order:

a.	FSC-A vs SSC-A to gate for live cells
b.	FSC-A vs FSC-H to gate for single cells in the forward direction
c.	SSC-A vs SSC-H to gate for single cells in the side direction
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
33. Create a graph that shows BFP fluorescence on the x-axis and mCherry on the y-axis.
 
Adjust the BFP laser setting if needed so that the main population (with strong BFP fluorescence) is centered over 10^5. 
Adjust the mCherry laser setting if needed so that the main population (high mCherry fluorescence) is centered over 10^5 on the y axis.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
34. Create a gate for the BFP-negative and mCherry-positive cells (in the top-left corner of the graph). This will be the sorted population. Note the percentage of the single cell population that expresses mCherry. Click on “Record data”. 
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
35. Create a graph from the BFP-negative, mCherry-positive cells that shows the ratio of GFP to mCherry on the x-axis and select “histogram” on the y-axis. 

This graph will set the gates for sorting the cells.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
36. Record data for all controls before beginning sorting.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
37. Load a library tube in the sample holder and record data for this tube.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
38. Create 4 gates on the GFP:mCherry graph - they should each contain ~25% of the total population shown on the graph. Label them Bin 1-4 from left to right.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
39. Install the recovery tubes in the sorter tray with Bin 1 on the left and Bin 4 on the right.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
40. In the software’s experiment dashboard, open the “Cytometer settings”, then open a sort layout. 
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
41. Choose the stall which contains the recovery tube for Bin 1, and select the gated population labeled as Bin 1 to sort into the recovery tube. Repeat for the remaining 3 bins.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
42. Click on the “Sort” button in the sort layout window.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
43. As the flow cytometer sorts the cells, pay attention to the volumes in both the original flow tube and the recovery tube. Do NOT let the original flow tube run empty, or the recovery tube overflow. To stop the sorter, click on “Pause” in the sort layout window - this does not stop the sorter from pulling from the original flow tube. Click on “Stop Acquisition” or “Unload” if it is necessary to change the tube on the sample loader. 

Record the number of cells sorted into each Bin for each set of recovery tubes to keep track of the total number of cells sorted.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
44. Sort cells into the prepared recovery tubes until the total number of cells needed is reached.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
45. Remove tubes from the cytometer and follow instructions for post-sorting cleanup given by the manager of the flow cytometer.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
46. Return the sorted cells to the tissue culture room. Centrifuge the recovery tubes at 300 x g, 5 min. Look for a small cell pellet at the bottom of the tubes.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
47. Aspirate the media from the recovery tubes, being very careful not to disturb the pellet.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
48. Resuspend the cells from each Bin in a total of 1mL D10 media. 

Ex: Combine all recovery tubes from Bin 1 by resuspending in 1mL divided between the recovery tubes and combining them in one tube after resuspension.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
49. Plate the cells from each sorted Bin into a T75 tissue culture flask with 9 mL D10 media (plus the 1mL of resuspended cells).

 

SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
50. Add 10 µL doxycycline to each flask and swirl to mix.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
51. Check there are cells in the flask by looking at each flask under the microscope.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
52. Place the T75 flasks into the incubator (5% CO2, 37 °C).

SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
53. Repeat steps 28-48 the next day for the remaining half of the cultures.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
54. Plate the cells sorted on this second day of sorting in the same flasks as the first day (e.g. Bin 1 from sorting day 2 is added to the Bin 1 flask from day 1 of sorting).
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
55. Incubate at 5% CO2, 37 °C until cells are 100% confluent.

 

SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
56. When cells are confluent, lift the cells in each flask.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
57. Centrifuge the cultures at 300 x g, 5 min. 
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
58. Aspirate the supernatant, do not disturb the pelleted cells.
SECTION: Fluorescence-activated cell sorting (FACS) - 2 days total
59. Freeze the pellets in labeled 15-mL centrifuge tubes until ready to extract genomic DNA from cells.