Pei max solution

Complete growth media was used for HEK293T cell culture. Complete growth media includes: 1:1 DMEM (Dublecco’s Modified Eagle medium, GIBCO): MEM (Modified Eagle medium, GIBCO), 10% FBS (Fetal Bovine Serum, Sigma), 1% (v/v) penicillin-streptomycin (Gibco).
Cells were plated at a density so that they would reach 80–90% confluence on the day of transfection. Cells were cultured at 37 °C under 5% CO₂.
For transfection, we pre-treated 48-well plastic plates with 200 μL of 20 μg/mL human fibronectin (Milipore Sigma) for 10 minutes at 37 °C under 5% CO₂. After 10 minutes, we removed the fibronectin and added 200 μL of 80–90% confluent HEK293T cells into each well. To prepare the transfection mixture for each well, we combined 100 ng of DNA with 1 μL of PEI MAX solution (polyethyleneimine, Polysciences) in 10 μL of DMEM and incubated at room temperature for 10 minutes. After the incubation, we added 100 μL of complete growth media to the DNA-PEI MAX mixture and mix it with the cell suspension in the well. Cells were incubated at 37 °C with 5% CO₂ until stimulation 20–24 hours later.

Lentiviral shRNA or sgRNA plasmids were constructed by inserting target oligonucleotides into pLKO.1 (Addgene, #10878) or lentiGuide-Puro (Addgene, #52963) plasmids, respectively. Plasmid DNA was extracted using a DNA extraction kit (Vazyme, DC112–01). The lentivirus was packaged by transfecting the plasmids with packaging vectors (psPAX and pMD2.G) and PEI MAX solution (Polysciences, #24765) into HEK293T cells. Afterward, the virus supernatant was collected, filtered with a 0.45 μm strainer, concentrated with PEG6000 (Sigma, #81253), resolved in PBS and then aliquoted for subsequent transfection. Cells were infected with viruses and selected for 72 hours with puromycin (0.5–2 μg/mL, Yeasen, 60210ES25), blasticidin (5–20 μg/mL, YEASEN, 60218ES60), or G418 (100–250 μg/mL, Yeasen, 60220ES03). The target oligonucleotides are listed in Supplemental Table 2.