Lipofectamine 3000

Primed and naive H9 ESCs were transfected using either Nucleofection (Lonza, VPH-5022), or using Lipofectamine 3000 according to manufacturer’s instructions. For each transfection, 6-10 million cells were used (approximately 2.5-4.2 x10⁸ cells in total) and transfected with 8 μg of plasmid library DNA and 500 ng pmCherry-N1 plasmid (Clonetech) as transfection control. Cells were incubated in 10 cm dishes and 24h post-transfection, single cells were harvested and subjected to FACS. Non-transfected cells were used to set sorting gates, DAPI was used as a marker for dead cells. All percentages mentioned are relative to the fraction of DAPI-negative, single cells.

HEK293T cells, HCT116 cells, and their derivatives were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and Penicillin-Streptomycin (Pen-Strep) at 37°C in a 5% CO₂ incubator. U2OS cells were cultured in RPMI media supplemented with FBS and Pen-Strep at 37°C in a 5% CO₂. HEK293T and HCT116 cell were transiently transfected using polyethylenimine MAX (PEI-MAX) according to the manufacturer’s protocols. U2OS cells were transiently transfected with Lipofectamine 3000 or LONZA according to manufacturer’s protocols.
Lentivirus was generated using pLenti-puro (7 ug), psPAX2 (5.25 ug), and pMD2.G (1.75 ug) plasmids that were transfected in LentiX-293T cells within 15 cm plates with PEI-MAX at a 4:1 (ug:ug) ratio with cDNA. Virus was produced for 48 hours in the media and collected, centrifuged, and filtered through a sterile 0.45 um filter (Cat. No. SE1M003M00). The virus containing medium was then supplemented with 10 ug/mL of polybrene and allowed to incubate with the targeted cells for 24 hours at 37°C in a 5% CO₂ incubator. The cells were washed and split for either chemical selection with puromycin or cell sorting.