Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cells were harvested by trypsinitation (Trypsin/EDTA solution, Thermo Fisher Scientific Inc.), collected by centrifugation (5 min at 300g), washed with ice-cold PBS with 100 µg/ml CHX added and recovered again by centrifugation. Cells were re-suspended in 1 ml ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM dithiothreitol (DTT), 100 µg/ml CHX, 1 × complete and EDTA-free protease inhibitor cocktail (Roche)), lysed by incubation on ice for 10 min and subsequently passed through QIAshredder spin columns (Qiagen). The flow-through was clarified by centrifugation for 10 min at 16,000 × g and 4°C. The supernatant was recovered and protein concentration was measured. For ribosome profiling (ribo-seq), 600 µl of each lysate was subjected to RNase I (Thermo Fisher Scientific Inc.) digestion using 3000 U of enzyme for every 7 mg protein. Digestion of polysomes proceeded for 45 min at room temperature and was stopped with SUPERase.In RNase Inhibitor (Life Technologies). Next, monosomes were recovered by ultracentrifugation at 75,000 RPM in a cooled TLA-120.2 rotor over a 1 M sucrose cushion in 20 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 100 µg/ml CHX and 100 U/ml Superase.In. RNA was extracted from the samples using a heated acid phenol; chloroform; isoamyl alcohol (125:24:1) procedure. For ribo-seq, ribosome protected fragments (RPFs) of 26-34 nucleotides were extracted from 22 µg of RNA subjected to electrophoresis under denaturing conditions in 15% TBE-Urea polyacrylamide gel (Life Technologies). Extraction from gel was performed in RNase-free water for 10 min at 70°C and precipitated with 1/10 volume of 3 M sodium acetate pH 5.2, 1.5 µl GlycoBlue (Life Technologies) and 1 volume of isopropanol overnight at − 80 °C. Following a dephosphorylation reaction (end-repair) with 10 U of T4 polynucleotide kinase (New England Biolabs), the RPFs were ligated to a 1.5 µg of Universal cloning linker using 200 U of T4 RNA ligase II (both New England Biolabs). The ligated product was size-selected using denaturing gel electrophoresis and purified from gel as described above. Subsequently, reverse transcription (RT) was performed with 200 U of SuperScript III (Life Technologies) according to (Ingolia et al, 2011), followed by a size-selection of the RT product (described above). Ribosomal RNA derived contaminants were depleted from reverse transcribed samples using a custom set of 20 biotynylated deoxyoligonucleotides. 100 pmol of mixed probes with 60ul MyOne Streptavidin C1 DynaBeads (Thermo Fisher Scientific Inc.) were used each time. Next, samples were subjected to circular ligation using 100 U of CircLigase (Illumina), diluted and amplified by PCR using Phusion polymerase (New England Biolabs) for 12 cycles of denaturation (10 s at 98°C), annealing (10 s of 65°C) and elongation (5 s at 72°C) using primer pairs compatible with the Illumina sequencing platform. The resulting ribosome-profiling libraries were duplexed (samples treated with the same inhibitor, LTM or CHX, were joined).