GSM3721666: LN308-6h-Tunicamycin-GAATGA-R1 (Ribo-Seq); Homo sapiens; OTHER
Ribo-Seq  TH- or TM-stimulated cells were treated with 0.1mg/ml cycloheximide for 5min.  After washing with ice-cold PBS (supplemented with cycloheximide), cells were harvested and lysed for 15min on ice in a buffer containing 5mM Tris pH7.4, 1.5mM KCl, 5mM MgCl2, 0.5% Triton-X, 0.5% sodium deoxycholate, 0.1mg/ml cycloheximide, RNasin and 1x cOmplete protease inhibitor cocktail (Roche).  The cell lysate was cleared by centrifugation and digested with 0.2U RNaseI (Ambion) per 1µg RNA for 15min.  To stop the reaction, 1U SUPERaseIn RNase Inhibitor (Invitrogen) was added per unit of RNaseI.  Ribosomal complexes were separated on 12ml 10-50% sucrose gradients (in a buffer containing 20mM Tris pH7.4, 75 mM NaCl, 5mM MgCl2, 0.1mg/ml cycloheximide, and 1mM DTT) by centrifugation at 4°C and 35,000rpm for 3h in a SW41 rotor.  Fractions were collected at 0.5ml/min with continuous monitoring of conductivity and UV absorption at 254nm.  Fractions containing 80S monosomes were diluted with an equal amount of RNase-free water followed by organic extraction with Phenol:Chloroform:Isoamylalkohol 25:24:1 (Roth) and ethanol precipitation.  RNAs were then separated by denaturing Urea-PAGE and visualized by SYBR Gold staining.  RNAs with a length of 27-33nt were excised from the gel and eluted overnight in a buffer containing 300mM NaOAc pH 5.5, 1mM EDTA, and 0.25% SDS followed by ethanol precipitation. RNA-Seq  Total cellular RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Ribo-Seq  After dephosphorylation for 1h at 37°C with 20U of T4 Polynucleotide Kinase (NEB), RNAs were ligated to a universal miRNA cloning linker (NEB) using truncated T4 RNA ligase 2.  After gelpurification reverse transcription was performed using SuperscriptIII reverse transcriptase and a primer complementary to the ligated adapter, followed by alkaline hydrolysis of the RNA strand.  The resulting cDNAs were gelpurified and contaminating sequences originating from rRNA were depleted by substractive hybridization to a mixture of biotinylated DNA oligonucleotides and Streptavidin magnetic beads (New England Biolabs).  The recovered RNAs were then circularized with CircLigase II ssDNA Ligase (Epicentre) according to the manufacturer's instructions and subjected to PCR amplification to generate amplicons suitable for Illumina sequencing.  After gelpurification, DNA concentration was determined using a Qubit Fluorometer (Qubit 2.0, Thermo Fisher Scientific).  Bioanalyzer or TapeStation (Agilent) analyses were performed to assess the quality of the samples followed by deep sequencing on the Illumina platform. RNA-Seq  RNA integrity was checked using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), and concentration was measured with Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies).  Starting with ~300ng of total RNA as input, ribosomal RNA was removed by NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs).  Subsequently, stranded total RNA-seq libraries were prepared from rRNA free RNA sample using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs) according to the protocol.  RNA-seq barcoded libraries that passed the QC step, which was assessed on the Agilent Bioanalyzer system, were then pooled in equimolar amounts;  1.8 pM solution of this pool was loaded on the Illumina sequencer NextSeq 500 and sequenced bi-directionally.
GEO Accession
GSM3721666