RNA from HEK cells was isolated using the DirectZol RNA MiniPrep Kit (Zymo) according to the manufacturer's protocol. cDNA was generated using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), and coding regions of RPS27A without the N‐terminal ubiquitin sequence was PCR amplified and cloned into a pcDNA5/FRT/TO vector backbone (Invitrogen) that had been previously modified to have a constitutive CMV promoter and C‐terminal SBP‐tag.
To generate stable transgenic cell lines, 1 × 106 HEK Flp‐In T‐Rex Cells (Invitrogen) were electroporated using a Lonza 4D Nucleofector in according to the Amaxa 4D‐Nucleofector™ Protocol for HEK293 (Lonza) for large cuvettes with 1.8 μg pOG44 Flp‐Recombinase Expression Vector (Invitrogen) and 0.2 μg pCMV‐RPS27A‐SBP. Two days after electroporation, cells were passaged and placed on media containing 5 μg·mL−1 blasticidin (Invitrogen) and 10 μg·mL−1 Hygromycin B (Thermo Fisher Scientific) until all cells from a control plate electroporated with pmaxGFP™ Vector (Lonza) were dead. Flp‐In cell lines were validated using anti‐SBP westerns and Sanger sequencing of the transgenic insert.
To generate stable transgenic cell lines, 1 × 106 HEK Flp‐In T‐Rex Cells (Invitrogen) were electroporated using a Lonza 4D Nucleofector in according to the Amaxa 4D‐Nucleofector™ Protocol for HEK293 (Lonza) for large cuvettes with 1.8 μg pOG44 Flp‐Recombinase Expression Vector (Invitrogen) and 0.2 μg pCMV‐RPS27A‐SBP. Two days after electroporation, cells were passaged and placed on media containing 5 μg·mL−1 blasticidin (Invitrogen) and 10 μg·mL−1 Hygromycin B (Thermo Fisher Scientific) until all cells from a control plate electroporated with pmaxGFP™ Vector (Lonza) were dead. Flp‐In cell lines were validated using anti‐SBP westerns and Sanger sequencing of the transgenic insert.