Zymopure plasmid dna isolation kit

Cell lines were generated as previously described [13 (link)]. In brief, SIM2 constructs were generated via long cDNA synthesis. Plasmids were amplified using Subcloning Efficiency™ DH5α™ competent cells (Life Technologies). Plasmid DNA was isolated using the HiPure Plasmid Maxiprep kit (Life Technologies) or the ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Ten micrograms of plasmid was mixed with GeneJuice (EMD Millipore) in 1 mL of Opti-MEM (Life Technologies) and incubated at room temperature for 15 min. This mixture was then added onto Phoenix-AMPHO lentiviral packaging cells (ATCC). Cells were incubated for 24 h at 32 °C and 5% CO₂. Media was collected and filtered through a 0.45-μm filter. The recommended amount of Sequabrene (Sigma) was added to the filtered media. The media was then added to SUM159 cells in six-well plates. Plates were centrifuged at 200×g for 60 min and allowed to incubate overnight at 32 °C and 5% CO₂. Media was again collected from the packaging cells the next day, and target cells were transduced a second time, as described above. Puromycin selection (2 μg/mL) was started the following day and maintained for at least a week [14 (link)].

SIM2s-FLAG SUM159 and shSIM2 MCF7 cells were generated as previously described^{36 (link),44 (link)}. The CRISPR/Cas9 SIM2 KO vector (pLV[2CRISPR]-hCas9:T2A:Puro-U6 > hSIM2[gRNA#1]-U6 > hSIM2[gRNA#2]) was designed with two gRNA sequences targeting exon 1 in hSIM2 (gRNA#1 5’-TCCGCGCTACTTCCTCTTCA-3’; gRNA#2 5’-ATGCGCGCCGTCTTCCCCGA-3’) and was generated and transformed into Stbl3 competent cells by VectorBuilder.
Plasmid DNA was isolated using a ZymoPURE Plasmid DNA Isolation Kit (Zymo Research). Lentiviral transduction was performed as previously described, and cells were selected with 2 μg/mL puromycin (Sigma‒Aldrich) for 7 days or after five passages.