title: GSM9264178: mrna_fluc_minU_n1m_a; Homo sapiens; RNA-Seq
library_name: GSM9264178
library_construction_protocol: Cells were scraped from 10-cm plates on ice and in the presence of 500ul lysis buffer (1% Triton X-100 in 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol supplemented with 10 U ml−1 Turbo DNase and 100 μg ml−1 CHX). After lysis, samples stood on ice for 20 min and then were centrifuged at 14,000 x g for 15 min to remove cell debris. Supernatants were split into two tubes - one for RNAseq and one for Ribo-Seq library preparation. For RNA-seq, cells were harvested with Tri-Reagent (Sigma-Aldrich), total RNA was extracted. poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen). RNA-seq: mRNA samples were subjected to DNAseI treatment and 3' dephosphorylation using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and T4 PNK (NEB) followed by 3' adaptor ligation using T4 ligase (NEB). The ligated products used for reverse transcription with SSIII (Invitrogen) for first strand cDNA synthesis. The cDNA products were 3' ligated with a second adaptor using T4 ligase and amplified for 8 cycles in a PCR for final library products of 200-300bp. or RNA-seq  libraries were prepared using NEbNext Ultra II directional RNA library prep kit. Ribo-seq: samples were treated with RNaseI for 45 min at room temperature followed by SUPERase-In (Invitrogen) quenching. Subsequent Ribo-seq library generation was performed/ In brief, digested samples were loaded onto a sucrose solution (34% sucrose, 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol and 100 μg ml−1 CHX) and spun for 1 h at 100,000 rpm in a TLA-110 rotor (Beckman) at 4 °C. Pellet was collected using TRI reagent (Sigma-Aldrich) and the RNA was extracted using chloroform phase separation. For size selection, 15 μg total RNA was separated on a 15% TBE-urea gel for 65 min, and 28–34 bp footprints were excised using 28 and 34 flanking RNA oligos, followed by RNA extraction and rRNA depletion (NebNext rRNA Depletion Kit v2) and the subsequent Ribo-seq protocol.
identifiers.namespace: GEO
identifiers.id: GSM9264178_r1