GFP dropout screening

Titrating virus

1.     Transfect guide RNAs into 293 cells according to the Ca/Po transfection method.

2.     Harvest viruses 48 hours after changing the media and freeze in 1ml aliquots.

3.     Transduce 200 ul of frozen viral supernatant into the A375 cas9 cell line (or any other cas9 cell line currently growing in incubator) plated on 12 well plates.

4.     After two days, change the media and allow one day for recovery.

5.     Passage the cells at the correct split ratio base on the cell type and allow to grow for 3 or 4 days (175ul trypsin+825ul media for passage).

6.      Measure percent GFP using the macsquant.  Adjust viral supernatant up or down to achieve a 20% to 30% GFP+ ratio.

sgRNA GFP dropout to identify cancer dependencies

1.     Transduce and select for stable expression of Cas9 in a cell line.

2.     Plate 50,000 cells (or an appropriate amount) per well onto a 12 well plate.   Use 1mL media.  Make sure to include at least 1 extra well as a no-GFP control.

3.     The next day, transduce cell with correct amount of viral supernatant.

4.     After two days, change the media.

5.     Passage the cells and take the first time point 3 to 5 days post transduction (3 days for fast growing cell lines, 4-5 days for slower lines).   To passage – 175ul trypsin, quench with 825ul media.  Split 1:3 for a slow growing cell line, split 1:6 for a fast-growing line. 

6.     For the MacsQuant reading, filter 500ul of the unused cell suspension into a FACS tube (blue cap), and measure percent GFP. 

7.     Repeat steps 5 and 6 every 3 or 4 days (depending on cellular proliferation) until a total of 15-20 days is reached.