design: Initial Test-RNA-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
library_construction_protocol: Cells were harvested and lysed by adding 400 µl Invitrogen™ TRIzol™ Reagent (Invitrogen; Cat# 15596018) to each well. Control, N-AID-UPF1 and C-AID-UPF1-tagged HCT116 cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). 3x10^5 cells were seeded in 24-well plates one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) at various time points during the experiment (between 0 and 12 hours before harvesting). All cells were harvested at the same time to minimize differences in cell numbers. Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions. poly(A) mRNA isolation and library preparation was carried out with 600 ng RNA as input using the NEBNext Poly (A) mRNA magnetic isolation module (NEB; Cat#E7490L) and NEBNext Ultra II Directional RNA library preparation kit (NEB; Cat#E7760L) according to the manufacturer's instructions.