GSM4594134: Ancestral FAS exon 6 Library Output 7b; Homo sapiens; OTHER
For each of the experimental replicates, 10 ng of the library were transfected into 250 000 HEK293 cells in one well of a 6-well plate using Lipofectamine 2000 (11668027, Thermo scientific) and OPTIMEM I Reduced Serum Medium (31985-047, Thermo scientific). Six hours post-transfection, the cell culture medium was replaced with DMEM Glutamax (61965059, Life Technologies) containing 10% FBS and Pen/Strep antibiotics. 48 hours post-transfection (72 in the case of the siRNA experiments), total RNA was isolated using the automated Maxwell LEV 16 simplyRNA tissue kit (AS1280, Promega). Accuprime Pfx (12344024, ThermoFisher Scientific) was used following the manufacturer's instructions to amplify 20 ng of single-stranded library DNA for 25 cycles with the following flanking intronic primers: FAS_i5_GC_F and FAS_i6_GC_R (Baeza-Centurion et al. 2019). The amplified library was then recombined with pCMV FAS wt minigene exon 5-6-7 (Förch et al. 2000) using the In-Fusion HD Cloning kit (639649, Clontech) in a 1:8 vector:insert optimized ratio and transformed into Stellar competent cells (636766, Clontech) to maximise the number of individual transformants (800,000 individual clones). The library was then amplified by growing for 18 hours in LB medium containing ampicillin. The final plasmid library were purified using the Quiagen plasmid maxi kit (50912163, Quiagen) and quantified with a NanoDrop spectrophotometer.
GEO
GSM4594134