GSM6008602: 1h 500 mOsm rep3; Homo sapiens; RNA-Seq
Ribosome profiling: Three 150 mm dishes of pRSV-T cells (80% confluency) were treated with hypertonic stress.  To avoid potential artifacts of pre-treating live cells with cycloheximide, it was added to the lysis buffer only after cells were harvested as previously described (PMID: 26657557, PMID: 30029003, PMID: 25056308, PMID: 31767846). After washing three times with ice-cold PBS, cells were scraped in 1 mL lysis buffer (10 mM HEPES pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton-X,100 μg/mL cycloheximide, 2 mM DTT, RNase inhibitor (1:100 dilution, NEB, M0314L), protease inhibitor (1 tablet per 10 mL, Roche, 04693159001). The lysate was passed four times through a 26G needle and centrifuged at 1300 x g for 10 min. Lysate (OD A260 = 10) was loaded on a linear 10%-50% sucrose gradient (gradient buffer is the same as the lysis buffer, lacking only Triton-X and Protease and RNase inhibitors) and centrifuged at 4°C at 31,000 rpm for 2.5 h using an SW 32 Ti rotor (Beckman Coulter, 369694). The gradients were fractionated using a Teledyne ISCO Density Gradient Fractionation System and 12 fractions were collected. The fractions from (including) the 80S peak to the bottom of the gradient were combined. 300 μL of the combined fractions was digested with 5 μL RNase I (Thermo Fisher Scientific, AM2295) for 1 h at 4°C (with slow rotation on a nutator). Immediately, Trizol LS (Thermo Fisher Scientific, 10296028) was added, and RNA isolated according to the manufacturer's instructions.  10 μg of total RNA isolated from the combined fractions was resolved by 15% TBE-Urea gel (Thermo Fisher Scientific, EC6885BOX) and visualized with SYBR™ Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, S11494); the 28-30 nt region was excised from the gel. The RNA was eluted in 400 μL RNA elution buffer (EB) at 4°C overnight (RNA EB: 0.3M sodium acetate (NaOAc, pH 5.5), 1 mM EDTA, 0.4 U/μL RNase inhibitor). Gel debris was removed using Corning Costar Spin-X centrifuge tube filters (Sigma-Aldrich, CLS8160). RNA was precipitated with 4 µL GlycoBlue Coprecipitant (Thermo Fisher Scientific, AM9515), 40 µL sodium acetate, and 1 mL ethanol overnight at -80°C. Recovered footprints were dephosphorylated in a 15 µL reaction mixture (1 µL T4 PNK (NEB, M0201S), 10X T4 PNK buffer, 0.4 U/μL RNase inhibitor) at 37°C for 1 h followed by heat inactivation at 65°C for 10 min. Dephosphorylated RNA was precipitated as described above and recovered in 4 µL of water. Pre-adenylated DNA linker (DNA oligo purchase from IDT was adenylated using 5´ DNA Adenylation Kit (NEB, E2610S)) was ligated to the 3'-end of RNA in a 20 µL reaction (25% PEG8000, 1X T4 RNA ligase buffer, 1 mM DTT, 0.4 U/μL murine RNase inhibitor, 300 U T4 RNA ligase truncated KQ (NEB, M0373S), 1 µM pre-adenylated DNA linker) for 6 h at 25°C followed by overnight ethanol precipitation as described above. Ligated footprints were resolved by 10% TBE-Urea Gel (Thermo Fisher Scientific, EC6875BOX), purified, eluted in the RNA EB at 4°C overnight, precipitated at -80°C as described above and resuspended in 10.5 µL water. 1 µL of 2.5 µM RT primer was added and heated at 65°C for 5 min, cooled on ice and incubated in 20 µL RT mix (300 U Superscript III (Thermo Fisher Scientific, 18080044), 1X RT buffer, 500 µM dNTPs, 5 mM DTT) for 30 min at 55°C, then heat inactivated at 70°C for 15 min.  RT product was precipitated with ethanol as described above, resuspended in 10 µL water and resolved by 10% TBE-Urea gel. cDNA was isolated and eluted overnight in DNA elution buffer (DNA EB: 0.3 M NaCl, 1 mM EDTA), precipitated overnight, recovered and used for the 20 µL circularization reaction (100 U CircLigase ssDNA Ligase (Lucigen, CL4111K), 50 mM ATP, 2.5 mM MnCl2, 1X Circligase buffer) at 60°C for 2 h. rRNA depletion was performed according to Ingolia et al. (Ingolia et al., 2012). Circularized, rRNA-depleted cDNA was used in the PCR using NEBNext Ultra II Q5 Master Mix (NEB, M0544S) and NEBNext Multiplex Oligos for Illumina (NEB, E7335S). Half of the cDNA was used to determine the optimal PCR cycle number. The PCR products were resolved by 8% TBE gel (Thermo Fisher Scientific, EC6215BOX). The other half of the cDNA was amplified by the determined PCR cycle, resolved by 8% TBE gel, and PCR products were cut, eluted in DNA EB, precipitated as described above, and recovered in 15 µL water. The quality and quantity of libraries were confirmed using DNA High Sensitivity Bioanalyzer (Agilent). Equal amounts of libraries were mixed and sequenced using SE75, NextSeq 500 (Illumina) sequencing platform.    RNA-seq: RNA was isolated from the cytoplasmic lysate (input that was loaded on the sucrose gradients for the abovementioned ribosome profiling) using Trizol LS according to the manufacturer's instructions, except that precipitation with isopropanol was conducted overnight at -80°C. 1 µg RNA was depleted from rRNA using NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB, E6310L) and was used to make libraries with NEBNext Ultra™ II Directional RNA Library Prep (NEB, E7765S) according to the manufacturer's instructions. The quality and quantity of libraries were confirmed using DNA High Sensitivity Bioanalyzer (Agilent) and sequenced using PE150, NovaSeq sequencing platform. RNA libraries were prepared for sequencing using standard Illumina protocols (see also extract protocol)