title: RNA-Seq of UPF1, SMG5 or SMG6 depletion in human embryonic kidney cell line HEK293 via the dTAG degron system and siRNA-mediated knockdowns
design: RNA-Seq of UPF1, SMG5 or SMG6 depletion in human embryonic kidney cell line HEK293 via the dTAG degron system and siRNA-mediated knockdowns
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). All cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). 2.8x10^5 cells were seeded in 6-well plates and reverse transfected with 2.5 µl Lipofectamine RNAiMAX (Invitrogen; Cat# 13778150) and 60 pmol of the respective siRNAs (Luciferase- or SMG5/SMG6-targeting) according to the manufacturer's protocol. Medium was changed the next day and 48 hours after knockdown, protein depletion was induced with 0.25 µM dTAGV-1 (Tocris Bioscience; Cat# 6914) for 24 hours, same volumes of DMSO served as controls. Thus, the cells were harvested 3 days after the initial knockdown.  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. ERCC RNA Spike-In Mix 1 (Thermo Fisher Scientific, Cat# 4456740) was added to the total RNA sample before library preparation. Libraries was prepared using the Stranded mRNA Preparation Kit (Illumina). 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. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (12 PCR cycles) to create the final cDNA library. After validation (TapeStation, Agilent Technologies) and quantification (Qubit, Thermo Fisher Scientific) individual libraries were pooled. 