title: RNA-Seq of H1299 cells transfected with different types of bi-directional plasmid to better understand the transcriptional changes occurring with the different design frameworks
design: RNA-Seq of H1299 cells transfected with different types of bi-directional plasmid to better understand the transcriptional changes occurring with the different design frameworks
library_construction_protocol: Transfections were carried out in 24-well plate for flow cytometry analysis.   H1299 cells were transfected with Lipofectamine® 3000 according to manufacturer's instructions and 300 ng of total DNA in 24-well plates. CHO and HEK293 were transfected with PEI transfection reagent with 300 ng of total DNA in 24-well plates. All cells were plated approximately 24h before transfection at 70000 to 80000 cells per well in 24-wells plates. At the moment of transfection, cells were put in starvation conditions, meaning in poor media for the first 24h. DNA was diluted in Opti-MEM reduced serum media (Gibco), before being mixed and incubated for 25 minutes prior to addition to the cells. All drugs were added 4h after transfection.  Modified-RNAs (modRNA) were transfected using Lipofectamine® 3000 protocol under modified conditions in which no P3000 reagent was used. RNA was first diluted in Opti-MEM reduced serum media (Gibco) and it was then mixed with diluted Lipofectamine reagent as manufacturer's instructions. The mix was incubated for 7 minutes prior to addition to the cells. The fast-forward protocol was used by seeding 120000 cells per well in 24-wells plates at the moment of transfection.  Libraries were prepared using QuantSeq 3' mRNA sequencing for RNA quantification kit (Lexogen)