design: RiboMinus 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 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). 2.5x10^5 cells were seeded in 6-well plates. 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 48 hours before harvesting). For the 48-hour time point, the medium was exchanged once for fresh medium with IAA. 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. Libraries were prepared from 500ng total RNA. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. Enzymatic depletion of ribosomal RNA with the Illumina Ribo-Zero Plus rRNA Depletion Kit was followed by library preparation with the Illumina® Stranded Total RNA sample preparation kit. The depleted RNA was fragmented and reverse transcribed with random hexamer primers, second strand synthesis with dUTPs was followed by A-tailing, adapter ligation and library amplification (12 cycles). Next library validation and quantification (Agilent Tape Station) was performed, followed by pooling of equimolar amounts of library.