title: GSM4715425: NRVCs treated with PE_Ribo_seq_rep1; Rattus norvegicus; OTHER
library_construction_protocol: For ribosome profiling from cell, samples were snap frozen in liquid nitrogen after washed with ice-cold PBS containing 100 µg/ml cycloheximide. Then one 10-cm dish of cells were harvested in 400 µL ice-cold mammalian polysome buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, with 1 mM DTT, 1% Triton X-100, 0.1% NP-40, 500U/mL RNase Inhibitor, 1× EDTA-free Complete protease inhibitors, 1U/µl DNase I and 100 µg/ml cycloheximide). After dispersal of the cells, the lysate was passed ten times through a 26 gauge needlet, cleared by centrifugation at 20,000g for 5 min. About 120 µl of the supernatant was digested with 3 µl of RNase I (Life Technologies, AM2294) for 45 min at room temperature. SUPERase•In RNase Inhibitor (Life Technologies, AM2694) was then added to stop the reaction. Resulting monosomes were purified with MicroSpin S-400 HR columns (GE  Healthcare, 27-5140-01). And the RNA was isolated from the flow-through (approximately 125 µl) using 1 mL Trizol. To enrich the RFPs (ribosome footprints) , the RNA samples was subjected to the Ribo-Zero Gold rRNA Removal Kit (Illumina, MRZG12324) to remove ribosomal RNA fragments and separated them on a 17% 7.5M Urea-PAGE gel. The size range from 26 nt to 30 nt, defined by loading with 14-30 ssRNA Ladder Marker (Takara, #3416), was cut out. For Ribosome profiling, the purified RNA was end-repaired and then processed with the in house small RNA sequencing protocol and converted into libraries. Brifely, the RNA fragments was lingated to a pre-adenylated DNA linkers and followed a 5' RNA adaptor. After reverse transctiption, the cDNA was amplified and sequenced with Illumina X ten platform.
attributes.tag: GEO Accession
attributes.value: GSM4715425