TITLE: FACS screening to detect regulators of ROS
AUTHORS: Megan Lee, Neal Bennett, Ken Nakamura

[DESCRIPTION]
This protocol describes an adapted CRISPRi- and FACS- based genomic screen to identify genetic modulators of ROS. By incubating cells with ROS dyes that are then sorted with flow cytometry and sequenced, this method allows for the identification of genes that impact ROS levels.


[STEPS]
SECTION: Culturing K562 Cells
1. Make K562 Media
450 mL RMPI-1640 media (UCSF Media Core)
50 mL 10% Fetal Bovine Serum
5 mL 0.2 M glutamine
5 mL PenStrep
SECTION: Culturing K562 Cells
2. Transduce lentivirus containing your sgRNA library at an MOI < 1, lower for smaller libraries. We recommend a 2 hour spinfection at 32 degrees Celsius, at 1000x g, in the presence of 8 µg/mL polybrene.

SECTION: Culturing K562 Cells
3. After at least two days, add 0.65 µg/mL puromycin for 5 days to select for cells expressing sgRNA.
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
4. Resuspend K562 cells in metabolic substrate, drug or antioxidant treatment depending on specific paradigm
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
4.1. For antioxidant or drug treatment, incubate cells with 0.1 µM MitoQ or 1 mM Trolox and appropriate control or vehicle treatments for 2 hours prior to cell sorting, or with 20 µM OHG, 10 µM Vidofludimus, or 0.5 µM Brequinar.
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
4.2. For metabolic substrate, resuspend cells in PBS with metabolic substrate for 30 min prior to cell sorting
Basal media conditions: 2% fetal bovine serum, 10 mM glucose, 5 mM pyruvate
Respiratory-only media conditions: 2% fetal bovine serum, 10 mM pyruvate, 10 mM 2-deoxyglucose
Glycolytic-only media conditions: 2% fetal bovine serum, 10 mM glucose, 5 µM oligomycin, 3 mM 2-deoxyglucose
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
5. Sort cells on flow cytometer.  
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
5.1. Set forward-scatter and side-scatter gates to isolate single cells.  

SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
5.2. Select for cells that express your CRISPRi sgRNA (for instance, by BFP+ signal). 
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
5.3. Set your gates to isolate cells that clearly separate from unstained cells.  If collecting cells for
screening and sequencing, within this gate, collect cell fractions that have the highest 25% and lowest 25% signal for your ROS dye.  Otherwise, take recordings of 10,000 cells.
SECTION: Fluorescence-Activated Cell Sorting (FACS) for ROS
6. If you are screening and sequencing, centrifuge collected cell fractions at 600xg and remove the supernatant. Cell pellets can be kept at -20 °C until further processing
SECTION: CRISPR pipeline
7. Isolate genomic DNA from cell pellets using the Macherey-Nagel NucleoSpin Tissue Mini kit.
SECTION: CRISPR pipeline
8. Amplify sgRNAs in genomic DNA and tag with sequencing adapters and barcodes by PCR 
1.5 µg undigested genomic DNA per PCR reaction (using Q5 HotStart High Fidelity Polymerase)
SECTION: CRISPR pipeline
9. Pool PCR product per sample, if multiple wells or tubes were needed.
SECTION: CRISPR pipeline
10. Separate PCR product from unincorporated primers and nucleotides using the Qiagen GeneRead Size Selection Kit.
SECTION: CRISPR pipeline
11. Measure purity and quality of PCR product with Agilent Bioanalyzer machine.  
SECTION: CRISPR pipeline
12. Sequence PCR products on DNA sequencer.