title: GSM7966622: HEK293T ΔYB-1ΔYB-3 + YB-3, RNA-Seq, rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were chilled on ice and washed with PBS + cycloheximide (100 µg/ml), lysed with buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide (Sigma-Aldrich), 25 U/ml TURBO DNase (Ambion). The lysate was divided into two parts for Ribo-Seq and RNA-Seq library preparation. Nuclease footprinting and ribosome recovery for Ribo-Seq library preparation were performed according to (McGlincy 2017, PMID: 28579404). The adapter for Ribo-Seq had a UMI-sequence (5 random nucleotides) for deduplication. Total RNA for RNA-Seq library preparation was isolated using TRIzol LS Reagent (Thermo Fisher Scientific). The rRNA depletion was performed using a RiboMinus™ Eukaryote Kit v2 (Thermo Fisher Scientific) according to the manufacturer's recommendations. RNA-Seq libraries were constructed using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB). Ribo-Seq libraries were obtained according to (McGlincy 2017, PMID: 28579404) using CircLigase II (Lucigen), custom adapter with UMI-sequence (5 random nucleotides), and NEBNext Multiplex Oligos for Illumina Index Primers Set (NEB).