TITLE: Guide RNA Library Transduction of Cas9 Cancer 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 whole-genome CRISPR screening of stably expressing Cas9 cancer cell lines in triplicate using the commercially available Kusuke Yusa v1.1 whole genome gRNA library. It can be adapted for other gRNA libraries, under the assumption that there is a BFP reporter in the gRNA library. </div><div class = "text-block">The protocol can be followed assuming the following is known:</div><div class = "text-block">- The number of cells to be transduced </div><div class = "text-block">- The number of days required for screening</div><div class = "text-block">- How many cells to maintain throughout the screen</div><div class = "text-block">- The number of cells required per pellet</div><div class = "text-block">- The required coverage of the library</div><div class = "text-block">This protocol takes 16 days.</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/ccmwbdtbp.png" /></div></div>

[STEPS]
?. [Day 1: Transduction]
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: Transduction]
Dilute the gRNA library if required, in complete media. The defrosted 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: Transduction]
Carry out each library transduction in triplicate. Prepare each replicate in a 250ml storage bottle as per Table 1 to transduce the cells at 100x coverage. Mix well, and seed into the appropriate labware. Clearly label each replicate (R1, R2, and R3).  ABCDE1ComponentsT150T525T8752x T875s2Cells33 x10^633 x10^633 x10^633 x10^63Polybrene (µL)8 µg/mL = 26.78 µg/mL = 93.338 µg/mL = 156.668 µg/mL = 313.324Volume of virus library (mL)(LT result x 15.6*) /1000(LT result x 54.7*) /1000(LT result x 91.1*) /1000(LT result x 182.3*) /10005Media (mL)33.33 – (virus volume + cell volume + polybrene)116.67 – (virus volume + cell volume + polybrene)194.4 – (virus volume + cell volume + polybrene)388.8 – (virus volume + cell volume + polybrene)6Total volume per flask (mL)33.33116.67194.4388.8
It is recommended to use a media dispensing pump to dispense volumes greater than 50ml.
It is recommended to select the flask size for 33 million cells to comfortably fit for each cell line (see guidelines for more information).
ABCDE1ComponentsT150T525T8752x T875s2Cells33 x10^633 x10^633 x10^633 x10^63Polybrene (µL)8 µg/mL = 26.78 µg/mL = 93.338 µg/mL = 156.668 µg/mL = 313.324Volume of virus library (mL)(LT result x 15.6*) /1000(LT result x 54.7*) /1000(LT result x 91.1*) /1000(LT result x 182.3*) /10005Media (mL)33.33 – (virus volume + cell volume + polybrene)116.67 – (virus volume + cell volume + polybrene)194.4 – (virus volume + cell volume + polybrene)388.8 – (virus volume + cell volume + polybrene)6Total volume per flask (mL)33.33116.67194.4388.8
?. [Day 1: Transduction]
Remove   of stock cell suspension and seed into two wells of a 6 well plate ( per well). Add  media to each well. Place plate in the incubator. One well will be fixed on day 4 with a sample of transduced cells to act as a negative control to identify the transduction efficiency.
1 mL
500 µl
1.5 mL
Maintain the second well for the remainder of the screen, passaging when required. This well will be fixed when puromycin selection efficiency is assessed to act as the negative control.
?. [Day 2]
Media change the flasks. Passage cells if required, keeping all cells (see protocol "Passaging adherent cancer cell lines", hyperlink in step 1).
It is recommended to use a media pump to dispense volumes greater than 50ml.
?. [Day 3]
Passage cells if required, keeping all cells (see protocol "Passaging adherent cancer cell lines", hyperlink in step 1).
?. [Day 4: Puromycin Selection]
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
?. [Day 4: Puromycin Selection]
Take an aliquot of ~200,000 cells in a 1.5ml tube to test transduction efficiency using FACS.
The transduction efficiency for the library needs to be >15% to ensure a sufficient level of library representation. It should be ~30% for each replicate.
?. [Day 4: Puromycin Selection]
Seed all remaining cells at a 1:3 split ratio (slow growing lines can be split 1:2) in appropriately sized labware (e.g.: if the cells were in 1x T875, re-seed all cells into 3x T875s) in the presence of puromycin at the concentration determined by the puromycin titration, see Table 2 for volumes.  ABCDEF1Size of FlaskVolume of Media (ml)1µg/ml Puromycin (µl)2µg/ml Puromycin (µl)3µg/ml Puromycin (µl)4µg/ml Puromycin (µl)2T52550601201802403T875100100200300400
It is recommended to use a media pump to dispense volumes greater than 50ml.Puromycin is toxic if swallowed and harmful in contact with skin.
ABCDEF1Size of FlaskVolume of Media (ml)1µg/ml Puromycin (µl)2µg/ml Puromycin (µl)3µg/ml Puromycin (µl)4µg/ml Puromycin (µl)2T52550601201802403T875100100200300400
?. [Day 4: Puromycin Selection]
Detach and collect the negative control cells from the 6 well plate following steps 8-11 in the protocol "Library Titration of Cas9 Cancer Cell Lines" found here: https://protocols.io/view/guide-rna-library-titration-of-cas9-cell-lines-bgxujxnw.html
?. [Day 4: Puromycin Selection]
Fix the 4 cell suspensions following protocol "Fixing cell pellets" (R1, R2, R3, negative control) found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
?. [Day 4: Puromycin Selection]
Carry out flow cytometry and FACS analysis following steps 16-20 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).
?. [Day 7-11]
Inspect cells under a microscope. If cells are
It is important that at least 50 million cells are retained at every passage going forward to maintain the integrity of the library as this maintains 5 times the original 100x coverage.
?. [Day 7-11]
If cells are >90% confluent, passage each replicate, and carry out FACS analysis to determine if puromycin selection has been successful:
?. [Day 7-11]
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
?. [Day 7-11]
Take an aliquot of ~200,000 cells in a 1.5ml tube per replicate.
?. [Day 7-11]
Fix the 4 cell suspensions following protocol "Fixing cell pellets" (R1, R2, R3, negative control) found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
?. [Day 7-11]
Carry out flow cytometry and FACS analysis following steps 16-20 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).
If puromycin selection has been successful, the proportion of BFP positive cells should be >80%
?. [Day 7-11]
If puromycin selection was successful, re-seed a minimum of 50 million cells per replicate into an appropriate number of flasks, following protocol "Passaging adherent cancer cell lines" (hyperlink in step 1) without puromycin. The screen can be continued in the absence of puromycin.
?. [Day 7-11]
If puromycin selection was unsuccessful, re-seed all cells into an appropriate number of flasks, following protocol "Passaging adherent cancer cell lines" (hyperlink in step 1) in the presence of puromycin.
7 days post puromycin selection, media change the cells without puromycin. The remainder of the screen can be continued in the absence of puromycin.
?. [Day 7-11]
Following puromycin selection, passage cells when >80% confluent, keeping a minimum of 50 million cells. If the cells are slow growing, keep a minimum of 100 million cells.
If by day 10, cells have not been passaged, media change the flasks without puromycin.If the cells do not require passaging until the day of pelleting, follow steps 11.2 - 11.4 to determine the puromycin selection efficiency on the day of pellet.
?. [Day 14/15/16: Pellet]
Pellet cells when confluent, between day 14 and day 16.Detach cells, collect, and count each replicate following protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
Collect the cell suspensions in PBS at step 7.
?. [Day 14/15/16: Pellet]
Aliquot 25 x106 cells into up to 6x 2ml PCR clean/DNA LoBind tubes and centrifuge at  for 5 minutes using a microcentrifuge in the MSC.
Centrifuge: 300 34
?. [Day 14/15/16: Pellet]
Carefully remove the supernatent using a P1000 pipette, ensuring not to dislodge the cell pellet. These cell pellets can be stored in a  freezer until DNA extraction is required.
-80 °C
?. [Day 7-11]
Detach and collect the negative control cells from the 6 well plate following steps 8-11 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).