Illumina NovaSeq 6000 paired end sequencing; mRNA-Seq from control cells and two clones of Flp-In-T-REx-293 cells modified via CRISPR-Cas9 to knockout SMG7 in combination to knockdowns of control, SMG5, and SMG6
mRNA-Seq from control cells and two clones of Flp-In-T-REx-293 cells modified via CRISPR-Cas9 to knockout SMG7 in combination to knockdowns of control, SMG5, and SMG6
Flp-In-T-REx-293 (human, female, embryonic kidney, epithelial; Thermo Fisher Scientific) cells were cultured in high-glucose, GlutaMAX DMEM (Gibco) supplemented with 9% fetal bovine serum (Gibco) and 1x Penicillin Streptomycin (Gibco). The cells were cultivated at 37°C and 5% CO2 in a humidified incubator. Generation of knockout cells using CRISPR-Cas9: The knockouts were performed using the Alt-R CRISPR-Cas9 system (Integrated DNA Technologies) and reverse transfection of a Cas9:guideRNA ribonucleoprotein complex using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol. The crRNA sequence (Design ID: Hs.Cas9.SMG7.1.AD; Integrated DNA Technologies) to target SMG7 was /AlTR1/rGrArArArArUrGrCrUrArGrUrUrArCrCrGrArUrUrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/. Reverse transfection was performed on 1.5x10^5 cells per crRNA in 12-well plates. 48 h after transfection the cells were trypsinized, counted and seeded at a mean density of a single cell per well in 96-well plates. Cell colonies originating from a single clone were then screened via Western blot and genome editing of SMG7 was analyzed on the genomic level via DNA extraction and Sanger sequencing. Alterations on the transcript level were analyzed via RNA extraction followed by reverse transcription and Sanger sequencing. siRNA-mediated knockdowns: Cells were seeded in 6-well plates at a density of 2- 3x10^5 cells per well and reverse transfected using 2.5 µl Lipofectamine RNAiMAX and 60 pmol of the respective siRNA(s) according to the manufacturer's instructions. siRNA were purchased from Integrated DNA Technologies and had the following targeting sequence: for Luciferase (Luc): 5′CGUACGCGGAAUACUUCGA-3′; for SMG5: 5′ GAAGGAAAUUGGUUGAUAC-3′; for SMG6: 5′ GGGUCACAGUGCUGAAGUA-3′. RNA was isolated using peqGOLD TriFast (VWR Peqlab) following the manufacturer's instructions. Following changes were made: Instead of 200 μl chloroform, 150 μl 1-Bromo-3-chloropropane (Molecular Research Center, Inc.) was used. RNA was resuspended in 20 μl RNase-free water. The library preparation was performed with the TrueSeq mRNA Stranded kit (Illumina). 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 is 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 to create the final cDNA libraries. After library validation and quantification (Agilent tape station), equimolar amounts of library were pooled.