RNA-Seq of SMG1-SMG8-SMG9 complex alterations in human osteosarcoma cell line U2OS
RNA-Seq of SMG1-SMG8-SMG9 complex alterations in human osteosarcoma cell line U2OS
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). 2.5x10^5 cells were seeded in 6-well plates and treated 2 days later for 24 hours with DMSO as control, 0.1 µM or 1 µM SMG1i. Thus, the cells were harvested 3 days after the seeding.  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. 