title: GSM4769298: Ribo-seq 700 mOSM 15min Recovery 15min, Replicate 2; Mus musculus; OTHER
library_construction_protocol: After the indicated treatments cytoplasmic extracts were prepared and used to construct Ribo-seq and RNA-seq libraries Three 15-cm plates of MEFs (80% confluency) were treated with the indicated intensity of hyperosmotic stress. Cells were washed three times with ice-cold PBS and 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 (NEB, 1:100 dilution), protease inhibitor (Roche, 1 tablet per 10 mL). The lysate was passed four times through a 26-gauge 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 was 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 h and 20 min using an SW 32Ti rotor. The gradients were fractionated using a Teledyne ISCO 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 (Ambion, AM2295) for 1 h at 4°C (slow rotation on the nutator). Immediately, Trizol LS (ThermoFisher Scientific, #10296010) 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, and visualized with SYBR gold; bands from 28-30 nt were isolated. The RNA was eluted in 400 μL RNA elution buffer (EB) at 4°C overnigt (RNA EB: 0.3M sodium acetate (NaOAc, pH5.5), 1 mM EDTA, 0.4 U/μL murine RNase inhibitor). RNA was precipitated with 4 µL GlycoBlue™ Coprecipitant (ThermoFisher 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 murine 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 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 (M0373, NEB), 1 µM pre-adenylated DNA linker) for 6 h at 25°C followed by precipitation as described above. Ligated footprints were resolved on 10% TBE-urea PAGE gel, purified, eluted in the RNA EB at 4°C overnight, precipitated at -80°C as described above, and finally resuspended in 10.5 µl water. 1 µl of 2.5 µM RT primers were added and heated at 65°C for 5 min, cooled on ice, and incubated in RT mix (1.5 µL Superscript III, 1X RT buffer, 250 µM dNTPs, DTT) for 30 min at 55°C. RT product was precipitated with ethanol as described above, resuspended in 10 µl water and 2X loading dye, and resolved in 10% TBE urea PAGE 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 PCR using NebNext Ultra II Q5 mastermix and NEBNext multiplex oligos for Illumina. Half of the cDNA was used to determine the optimal PCR cycle number. The PCR products were resolved on a native 8% TBE gel. The other half of the cDNA was amplified by the determined PCR cycle, resolved on native 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 an Advanced Analytical Fragment Analyzer (Agilent). Equal amounts of libraries were mixed and sequenced using NextSeq500 (aiming for 40 million reads per library).
attributes.tag: GEO Accession
attributes.value: GSM4769298