alias: GSM2734594
title: GSM2734594: rSF+q1; Homo sapiens; RNA-Seq
library_construction_protocol: Ribosome footprint was performed according to (Ingolia et al, 2011). 2x107 HeLa cells from each culture condition were treated with cycloheximide (100 µg/ml) for 5 minutes prior to lysis in 800 µl of 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl, 1% Triton X-100, 100 µg/ml cycloheximide, 1×Complete Protease Inhibitors (Roche). 200 µl aliquot was used to extract cytoplasmic RNA to perform RNAseq.  200 µl aliquot was used to produce ribosome profiling. The left over 400 µl aliquot of lysate was treated with 4 U DNase I (ThermoScientific) and 800 U of RNase I (Ambion), for 45 minutes at room temperature with gentle shaking.   800 U of RNasin ribonuclease inhibitor (Promega) was added to quench the reaction.    The  samples were run on a 17.5-50% sucrose gradient to isolate monosome.  Footprint monosome RNA was end using RNA were end-repaired with T4 polynucleotide kinase (TaKaRa), and size selected 28-31 nucleotide on a 15% polyacrylamide TBE-urea gel. Small RNA libraries were performed according to the protocol using NEB NEXT Small RNA library Prep Set for Illumina (Multiplex Compatible) E7330, and size-selected (120-170 base pairs) before sequencing. RNA Sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the manufacturer's protocol. Briefly, poly(A)+ RNA was purified from 1µg of total RNA using oligo(dT) beads, fragmented and converted to cDNA. The double stranded cDNA fragments were then end-repaired, adenylated on the 3′ end, adapter ligated and amplified with 12 cycles of PCR. The final libraries were validated using Qubit Fluorometer (Life Technologies-Invitrogen) and Agilent TapeStation 4200 (Agilent Technologies). After sizing analysis, multiplexed sequencing libraries were normalized, pooled and clustered on the cBot (Illumina, Inc.) with a final concentration of 10 pM (spiked with 1% PhiX control v3, Cat No. FC-110-3001) using the TruSeq SR Cluster Kit v3 (Cat no. GD-401-3001). Sequencing on HiSeq 2000 (Illumina) was performed using the 50 cycles TruSeq SBS Kit v3 according to the manufacturer's protocol.
identifiers.namespace: GEO
identifiers.id: GSM2734594