TITLE: Guide RNA Library Titration of Cas9 Cell Lines
AUTHORS: Verity Goodwin, Emily Souster, Charlotte Beaver, Adam Jackson, Rizwan Ansari, Mathew Garnett, Fiona Behan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for the gRNA library titration of cas9 expressing cancer cell lines using the Kusuke Yusa v1.1 whole genome gRNA library. gRNA library titration is performed on all the cas9 cancer cell lines prior to gRNA library screening. The library titration allows us to determine the volume of library virus required to transduce cas9 cancer cells at 30% transduction efficiency which is calculated using FACS analysis.</div><div class = "text-block"><span style = "font-weight:bold;">Process diagram:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/11f7e279594fdb8947a627a79a011af77912ade8923993cae1dcb9e618960e5f/ccmjbdtbp.png" /></div></div>

[STEPS]
?. [Day 1]
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 found here: https://protocols.io/view/passaging-adherent-cancer-cell-lines-bgtbjwin.html
?. [Day 1]
Prepare a cell suspension containing 1.25x106 cells in  complete media to make a final concentration of 1.56x106 cells per ml.
8 mL
?. [Day 1]
Dilute the gRNA library in complete media if required. The defrosted gRNA library should be stored on ice and used within 1 hour.
Lentivirus: Lentiviruses used in this protocol can infect human cells but are non-replicating and therefore the pathogenicity of these viruses is negligible. Correst use of PPE will drastically reduce the risks.
?. [Day 1]
Add the diluted gRNA library volumes in Table 1 to the 6 well plate according to the layout in Figure 1. Mix well by rocking the plate gently.
?. [Day 1]
Add  of polybrene (a concentration of 8µg/ml) to the cell suspension and mix well using a 10ml stripette and pipetboy.
6.4 µl
?. [Day 1]
Transfer   of the cell suspension into 4 wells of a 6 well plate (this equates to 2.5x105 cells per well) and add the relevant media volume, see table 1, column 4.  ABC1Titration Volume (µl)Cell + polybrene suspension (ml)Volume of media (ml)201.64003151.63854501.635051501.6250
1.6 mL
ABC1Titration Volume (µl)Cell + polybrene suspension (ml)Volume of media (ml)201.64003151.63854501.635051501.6250
?. [Day 1]
Place the plate in the incubator.
?. [Day 2]
Carefully replace media with   fresh media per well, without dislodging the cells.
2 mL
?. [Day 4: FIxing]
?. [Day 4: Fixing]
Aspirate culture media from each well, and carefully wash each well with   PBS by angling the pipette at the side of the wells. Aspirate PBS.
2 mL
Day of harvest may be adjusted if cell lines take longer to express BFP. Some optimisation may be required.
?. [Day 4: Fixing]
Add   TrypLE to each well and place in the incubator for 3-5 minutes. Use the microscope if necessary to check that all the adherent cells have been dislodged. If not, return them to the incubator for a few more minutes until detached.
500 µl
?. [Day 4: Fixing]
When cells have detached from the base of the well, add  complete media to each well. Mix by pietting up and down using a P1000, and wash the base of the well ensuring cells are fully detached.
500 µl
?. [Day 4: Fixing]
Collect the 1ml cell suspension in a labelled 1.5ml centrifuge tube and centrifuge . Carefully aspirate the supernatent without dislodging the pellet.
Centrifuge: 300 34, 3 min
Centrifuge using a minifuge inside the MSC.
?. [Flow Cytometry and analysis]
Set up an experiment ensuring parameters: FSC-A, FSC-W, SSC-A, 450/50-A (BFP) are selected.
The following steps are for use on the BD LSRFortessa flow cytometer. It can be adapted for use on other flow cytometers.
?. [Flow Cytometry and analysis]
Run the mock sample (0) first and use this to separate debris (FSC-A vs SSC-A) and identify single cells (FSC-A vs FSC-W). Use the polygon gate to select for these cells (see Figure 2, top panel). If required, adjust FSC and SSC voltages so that the main population lies within the plot.
FSC will need to be reduced for very large cell lines.
?. [Flow Cytometry and analysis]
Check that the mock cells are negative for BFP using 450/50 (405)-A vs SSC-A and insert a polygon gate with the negative population positioned to the left of the gate, see Figure 2, bottom left panel. Run and record 10,000 events per sample.
?. [Flow Cytometry and analysis]
Next run the titrated samples, starting with the lowest volume of virus (15µl). The BFP positive cells should appear in the BFP+ gate, shown in Figure 2, bottom right panel. Run and record 10,000 events per sample.
Library infectivity is defined as the percentage of single cells that are BFP positive.
?. [Flow Cytometry and analysis]
Export FCS files. Carry out analysis using appropriate analysis software, such as FlowJo. Analysis and gating protocol are the same used to set up the flow cytometer as shown in Figure 2.
?. [Flow Cytometry and analysis]
Export data as the percentage of single cells that are BFP positive as an excel file for each titration.
?. [Flow Cytometry and analysis]
Plot a titration curve in Microsoft Excel by plotting the percentage of BFP% cells at each virus concentration, to calculate the amount of virus required for 35% transduction efficiency based on the linear portion of the curve. See Figure 3.
Although the transduction efficiency of 30% is ideal, we aim for 35% when scaling up to account for errors when increasing the surface area from a 6 well plate to a flask. For example, in Figure 3, the equation to determine the amount of virus that infects 35% of cells in one well of a 6 well plate is (35-11.324)/0.5057= 48.8µl. This value can then be scaled up to the desired surface area for transduction.
?. [Day 4: Fixing]
Follow "Fixing Cell Pellets" protocol found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html