design: RNA-Seq of EIF4A3 knockdown in human embryonic kidney cell line HEK293 via siRNA
library_construction_protocol: Cells were harvested with 350 µl lysis buffer from the NucleoSpin RNA Plus kit (Macherey&Nagel; Cat# 740984.50). The HEK293 cell line was maintained at 37°C and 5% CO2 in a humidified incubator 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). 2x10^5 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 EIF4A3-targeting) according to the manufacturer's protocol. Medium was changed the next day and cells were harvested 3 days after the initial knockdown. Total RNA was extracted with the NucleoSpin RNA Plus kit (Macherey-Nagel; Cat# 740984.50) according to the manufacturer's instructions. The Lexogen SIRV Set1 Spike-In Control Mix that provides a set of external RNA controls was added to the total RNA to enable performance assessment. Using the Illumina TrueSeq Stranded Total RNA kit library preparation was accomplished. This included removal of ribosomal RNA via biotinylated target-specific oligos combined with Ribo-Zero gold rRNA removal beads from 1 μg total RNA input. After depletion of cytoplasmic and mitochondrial rRNA, the remaining RNA 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 to create the final cDNA libraries. Next library validation and quantification (Agilent tape station) were performed, followed by pooling of equimolar amounts of library.