design: RNA-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 or human embryonic kidney cell line HEK293 via the dTAG degron 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 FKBP-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). For control and FKBP-UPF1-tagged HEK293 cell lines, DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059) was used and supplemented as described above. 5x10^5 cells were seeded in 6-well plates one day before starting the depletion experiment. UPF1 depletion was induced with 0.25 µM dTAGV-1 (Tocris Bioscience; Cat# 6914) for 12 hours. 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 with the Illumina® Stranded mRNA Preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. After poly-A selection (using Oligo(dT) magnetic beads), mRNA was purified, 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) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of library were pooled.