alias: GSM2735373
title: GSM2735373: control mouse brain, RiboLace 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. For RiboLace Ribo-Seq, the RNA absorbance of the lysate (at 260 nm) was measured by Nanodrop ND-1000 UV-VIS Spectrophotometer and the lysate diluted to 1.7 a.u. A260/µL. ARTseq nuclease was used to footprint ribosomes for 45 min at 20°C. The reaction was added directly to the RiboLace beads and the suspension incubated on a wheel for at least 1 hour in orbital rotation, 3 rpm at 4°C. After incubation, the tubes were kept on ice on a magnetic stand for 5 min to capture the beads–bounded-ribosomes. The supernatant was discarded (unbound fraction) and beads washed two times with 500 µL of a buffer. 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: GSM2735373