design: Ribo-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
library_construction_protocol: The cells were washed with PBS containing 100 μg ml−1 cycloheximide (Biochemika) before cell lysis. Samples were lysed in polysome buffer (100 mM Tris-HCl pH 7.4, 750 mM NaCl, 25 mM MgCl2, 1 mM dithiothreitol (DTT), 1% Triton X-100, 100 μg ml−1 cycloheximide and 25 U ml−1 Turbo DNase (Thermo Fisher Scientific)) and directly frozen in liquid nitrogen. 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 (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). 3x10^6 cells were seeded a 10 cm dish one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) for 12 hours. All cells were harvested at the same time to minimize differences in cell numbers. RNA was then digested with 2400 U ml−1 RNase I (Ambion) for 45 min in slow agitation at RT and digestion stopped with 640 U ml−1 SUPERase•In (Thermo Fisher Scientific). Monosomes were purified by size exclusion with Illustra MicroSpin S-400 HR columns (GE Healthcare) and extracted with 3 volumes of Trizol LS (Thermo Fisher Scientific), chloroform and the RNA Clean & Concentrator kit (Zymo- Research). 5000 ng of the isolated monosomes were depleted of rRNA using a RiboPool kit for Ribosome Profiling (siTOOLs Biotech). The RNA was separated on a 17% Urea PAA gel, 27–30 nt RNA fragments were eluted from the gel and 5' extremity phosphorylated with 10 U T4 polynucleotide kinase (New England Biolabs) for 1 h at 37 °C. For library generation, the NextFlex small RNA sequencing kit (PerkinElmer) was used according to the manufacturer's instructions.