design: SLAM-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 1 ml of in-house prepared TRI reagent to each well (prepared following DOI: 10.1371/journal.pbio.3000107). Both control and N-AID-UPF1-tagged HCT116 cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium with GlutaMAX supplement (Gibco; Cat# 36600088), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). 5x10^5 cells were seeded in 6-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 12 and 24 hours before harvesting). Except unlabeled N-AID-UPF1 controls, all cells were labeled with 200 µM 4-thiouridine (4SU; Abcam; Cat# ab143718) for 2 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. Thiol modification was performed as previously described by Herzog et al. 2017. Shortly, we mixed 1 µg of RNA with 10 mM Iodoacetamide (IAA; Sigma-Aldrich; Cat#I6125), 50 mM NaPO4 (pH 8) and 50 % DMSO. The reaction was performed at 50°C for 15 minutes, protected from light. Alkylation was quenched with 20mM DTT. RNA was precipitated by adding 2.5 volumes of 100% EtOH and 1 µg GlycoBlue (Ambion; Cat# AM9515) and incubating -80°C for 30 minutes. After cold centrifugation at 16,000 x g for 30 minutes, the pellet was washed with 75% EtOH, re-centrifuged for 10 minutes, air-dried for 5-10 minutes, and resuspended in 5-10 µl of H2O. poly(A) mRNA isolation and library preparation was carried out with 500 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.