RNA-Seq of UPF1 recovery in human colorectal adenocarcinoma cell line HCT116 previously depleted via the auxin-inducible degron (AID) system
RNA-Seq of UPF1 recovery in human colorectal adenocarcinoma cell line HCT116 previously depleted via the auxin-inducible degron (AID) system
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). 1.25x10^5 cells were seeded in 6-well plates. UPF1 depletion was induced for 24 hours with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148). Afterwards, the cells were incubated in medium without IAA for various time points (between 0 and 48 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. Libraries were prepared using the Illumina Stranded TruSeq RNA sample preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. Library preparation started with 500ng total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (15 cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of library were pooled.