alias: GSM2734702
title: GSM2734702: control mouse brain, poly-RIBOseq, P5, rep 2; Mus musculus; OTHER
library_construction_protocol: Cytoplasmic lysates were prepared pulverizing frozen mouse brains in a pestle and mortar cooled by liquid nitrogen and dissolving the powder in lysis buffer. Lysates were clarified by two following centrifugations. The lysates were loaded in a 15-50% linear sucrose gradient and polysomes separated by ultracentrifugation. Polysomal fractions were pooled and digested with Rnase I for 2h at 4°C. Ribosome Protected Fragments (RPFs) were extracted with acid-phenol:chloroform:isoamylalcohol and isopropanol precipitation and size selected (25-35 nt) by UREA Page. RPFs were treated to remove 3’ phosphate, ligated to the 3’ adaptor and purified by UREA page. Ligated RPFs were retrotranscribed and the cDNA circularized. Libraries were finally amplified by PCR using Illumina PCR Primer Index. Libraries were sequenced on an Illumina HiSeq2500.
identifiers.namespace: GEO
identifiers.id: GSM2734702