Materials #

Equipment

1. Electroporator
2. Mammalian cell incubator
3. Bacterial cell incubator with shaking
4. Thermocycler
5. Gel electrophoresis box
6. Bioanalyzer
7. Illumina sequencer
8. Flow cytometer with filters for BFP (Ex: 380/20, Em: 460/40)

Disposables

1. Sterile filtered pipette tips
2. 1.5 mL microcentrifuge tubes (sterile)
3. 1.8 mL Screw top cryovials (sterile)
4. 20 mL Luer-tapered syringe (sterile)
5. 0.45 $\mu$m polyethersulfone (PES) syringe filter
6. 30,000 molecular weight cutoff (MWCO) PES concentrator capable of processing 20 mL

Biologics

1. Electrocompetent e. coli suitable for unstable DNA (restriction minus, endonuclease deficient, and recombination deficient)
2. Cells of interest ¹

Plasmids

1. CROPseq gRNA expression transfer vector, Cropseq-BFP-WPRE-TS-hU6-BsmbI {Addgene # 137993 / Brock Lab AA112}
2. 10X Capture gRNA expression transfer vector, pLKV2-hU6-BbsI-PGK-Puro-TagBFP-WPRE {Addgene # 137994 / Brock Lab AA174}
3. Lentiviral packaging plasmid, VSV-G (Addgene #14888)
4. Lentiviral packaging plasmid, psPAX2 (Addgene #12260)
5. dCas9-VPR (Addgene #63798)
6. Recall-miniCMV-sfGFP (Addgene #137995 / Brock Lab #AA158)

Primers

(see Table 1)

Buffers

1. Buffer 3.1: 100 mM NaCl , 50 mM Tris-HCl, 10 mM MgCl₂, 100 $\mu$g/ml BSA , pH 7.9 at 25°C
2. NEB 5X Q5 Reaction Buffer
3. 10X T4 PNK Buffer
4. 10 mM dNTPs
5. 1X Tris-acetate-EDTA (TAE)
6. FACS Buffer: 5% FBS, 1-5 mM EDTA, 95% Phosphate-Buffered Saline

Enzymes

1. BsmBI (10,000 U/mL)
2. BbsI (10,000 U/mL)
3. NEB Q5 polymerase
4. T4 ligase (400,000 U/mL )
5. T7 ligase (3,000,000 U/mL)
6. PNK (10,000 U/mL)

Other Reagents

1. Lipofectamine^TM 2000
2. Lipofectamine^TM 3000
3. Nuclease-free water
4. Agarose
5. DNA Clean and Concentrator kit
6. 2xYT microbial growth medium
7. Dulbecco’s Modified Eagle Medium (DMEM)
8. OptiMEM^TM reduced serum medium
9. Fetal Bovine Serum (FBS)
10. Carbenicillin
11. Solid Phase Reversible Immobilization (SPRI) paramagnetic beads for PCR cleanup
12. 70% molecular biology grade ethanol in nuclease-free water
13. 10 mg/mL hexadimethrine bromide
14. 0.05% Trypan blue
15. Plasmid Midi-Prep Kit
16. DNA gel purification kit

Computational

1. Linux Machine
2. Python 3.8
3. Cell Ranger (for 10X analysis)
4. Pycashier