title: GSM1609377: GM10847_Rep1; Homo sapiens; OTHER
library_construction_protocol: For ribosome profiling experiments, a 34% (Weight/Volume) sucrose cushion was used. 7 A260 Units of the cleared cell lysate were incubated with 300 Units of RNase T1 (Fermentas) and 500ng of RNaseA (Ambion) for 30 minutes at room temperature. RNase digestion was stopped with 20 mM Ribonucleoside Vanadyl Complex (NEB: S1402S). No RNase control included 20 mM Ribonucleoside Vanadyl Complex in cell lysis buffer to inhibit any endogenous RNase activity. The sample was ultracentrifuged in a TLA120.3 rotor at 4ºC for 4 h at 70,000 rpm. For ribosome profiling experiments, the pellet was resuspended in 700ul of Qiazol reagent from miRNeasy kit. The RNA was purified using manufacturer’s instructions. Purified RNA was 3’end dephosphorylated using 10U of T4 PNK (NEB) at 37C for 3 hours. Dephosphorylated RNA was heated to 80C for 90 seconds before loading onto 15% (Weight/Volume) polyacrylamide TBE-Urea gel that was prerun at 180V for 15min. Electrophoresis was carried out for ~1.5hr at 180V. Custom RNA oligos were used to determine the 26-34nt size range on the gel and was excised on a DarkReader Transilluminator (Clare Chemical Research). Excised gel pieces were placed in GeBAflex electroelution tubes with 8 kDA molecular weight cutoff (Gerard Biotech). Electroelution was carried out at 120V for 90min, and the eluate was filtered through Spin-X 0.22um Cellulose Acetate centrifuge filter tubes (Sigma). 300 mM sodium acetate pH 5.5, 10 mM MgCl2, and 22.5 μg of GlycoBlue (Life Technologies) was added to filtrate. Equal volume of 100% isopropanol was used for precipitation at -20°C overnight. RNA was pelleted by centrifugation at 21000g at 4C. RNA pellet was washed once with cold 75% ethanol and resuspended in 6ul of nuclease free water. NEBNext Small RNA Library preparation kit was used to convert the isolated RNA fragments into Illumina sequencing library using manufacturer’s instructions except for the choice of 30C for 3hrs for all ligation reactions.
attributes.tag: GEO Accession
attributes.value: GSM1609377