title: GSM2724039: NAT10_KO_Ribo-seq_BioRep_2 (protected RNA); Homo sapiens; OTHER
library_construction_protocol: Total RNA was purified from cultured cells using Trizol (ThermoFisher) followed by treatment with TurboTM DNase (ThermoFisher). Enrichment of polyadenylated RNA (poly(A)-RNA) from DNase-treated total RNA was carried out by two rounds of selection with Oligo-(dT)25 Dynabeads (ThermoFisher) according to the manufacturer’s instructions. Enrichment efficiencies were estimated with Bioanalyzer picoRNA chips (Agilent Technologies, Santa Clara, CA). Parental and NAT10-/- cells were grown in complete DMEM medium to 80% confluency in three 10 cm plates per sample per replicate. Following quick media aspiration, cells were flash frozen by placing the plates on top of liquid nitrogen and immediately transferred to ice. Cells for each sample were scraped on wet ice into 500 μL lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, and 100 μg/mL cycloheximide), which was transferred from one plate to the next to collect the cells from all three plates for each sample. Cells were next triturated 10 times using a 26-gauge needle and the lysates were clarified by centrifugation at 20,000 X g for 10 min at 4°C. For ribosome profiling, 300-450 μL of each lysate were treated with 0.3 U/μL RNase I (ThermoFisher) for 40 min at room temperature, while the remaining lysate was flash frozen in liquid nitrogen for later total RNA isolation. Following the RNase I digestion, 5 μL of Superase-In (ThermoFisher) were added to each sample, and the samples were loaded onto 15-45% w/w sucrose gradients which were prepared, centrifuged, and fractionated as described in (J.E. Smith et al., 2014). Ribosome footprint RNA was isolated from fractions containing 80S monosomes and ribosome profiling libraries were prepared, as described previously, with a few modifications (J.E. Smith et al., 2014).  Briefly, RNA was isolated using two phenol/chloroform/LET (25 mM Tris-HCl, pH 8.0, 100 mM LiCl, 20 mM EDTA, pH 8.0) extractions, and 10 μg was depleted of ribosomal RNA only once, using the human/mouse/rat Ribo-Zero Gold rRNA removal kit (Illumina MRZG12324). For the total RNA fragmented controls, 200 μL LET was added to each 50 μL aliquot of lysate, and total RNA was extracted once with phenol/LET, once with phenol/chloroform/LET, and once with chloroform. Following ethanol precipitation, the total RNA was DNase I treated (Sigma-Aldrich). An RNA Spike-In mix (Thermo Fisher Scientific 4456740) was added to 5 μg of DNase I-treated total RNA according to the manufacturer’s instructions, and ribosomal RNA was depleted using the human/mouse/rat Ribo-Zero Gold rRNA removal kit. Next, the total RNA samples were fragmented in alkaline fragmentation buffer (1 mM EDTA, 50 mM Na2CO3, 50 mM NaHCO3, [pH 9.2] for 40 min at 95°C, as described previously (N.T. Ingolia et al., 2010), and fragments between 26 and 34 nucleotides in size were gel purified and used for library preparation in parallel with the ribosome profiling samples as described above (N.T. Ingolia et al., 2010). Illumina libraries were constructed using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®.
attributes.tag: GEO Accession
attributes.value: GSM2724039