GSM3109237: Input CMTR1 #2; Homo sapiens; RNA-Seq
Cells were incubated in 100 μg/ml cycloheximide for 10 min, washed in ice cold PBS supplemented with 100 μg/ml cycloheximide and extracts collected in polysome lysis buffer (15mM Tris (pH7.5), 15mM MgCl2, 0.3 M NaCl, 1 mM DTT, 1% Triton X-100, 100 mg/ml cycloheximide, 100 U/ml RNasin). 10% of the extracts were retained as input and 90% resolved by centrifugation through a 10 ml 10%–50% sucrose gradient at 18 000 x g for 2 hr at 4oC. Fractions were collected on FoxyR1 fractionator (Teledyne ISCO) with OD259 nm monitoring.  Polysomal fractions 16-20 were purified by Phenol:Chloroform:Isoamyl Alcohol (25:24:1). RNA was precipitated overnight with 2M of LiCl, 20mM Tris pH 7.5, EDTA 10mM. Input RNA was purified using Trizol Reagent. RNA was quality checked using a TapeStation (Agilent Technologies). TruSeq Stranded Total RNA with Ribo-Zero-Gold kit (Illumina).  Sequencing was performed using NextSeq Series High Output Kit 2 × 75 bp (Illumina).
GEO Accession
GSM3109237