design: RNA-Seq of SMG1-SMG8-SMG9 complex alterations in human colorectal adenocarcinoma cell line HCT116
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). HCT-116 (human, male, colorectal carcinoma, epithelial; ATCC, cat. no. CCL-247; RRID:CVCL_0291) and Flp-In-T-REx-293 (human, female, embryonic kidney, epithelial; Thermo Fisher Scientific, cat. no. R78007; RRID:CVCL_U427) were cultivated in high glucose, GlutaMAX DMEM (Gibco; Cat# 61965-026) supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). The cells were cultured at 37 °C and 5% CO2 in a humidified incubator. 3x10^5 cells were seeded in 6-well plates. After 24h, different final concentrations (0, 0.1 or 1 μM) of SMG1 inhibitor (SMG1i; DOI: 10.1016/j.bmcl.2012.08.107) dissolved in DMSO were added. After 24 h cells were harvested. 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.