alias: GSM1675585
title: GSM1675585: MB197_2; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen brain tissue was homogenized in Trizol (Invitrogen/Life Technologies, Carlsbad, CA), and then phase separated with chloroform. The RNA containing aqueous layer was diluted in binding buffer and applied to Qiagen (Venlo, Limburg, Netherlands) or Denville (South Plainfield, NJ) RNA isolation columns. The bound RNA was DNase treated and eluted according to kit procedures. Fifty nanograms of total RNA was converted to cDNA and isothermally amplified using the NuGen Ovation RNA-Seq kit procedures (NuGen, San Carlos, CA). The NuGen brain cDNA was sheared to approximately 150 bp fragments using the Covaris S (Woburn, MA). After shearing, fragments were recovered by centrifuging over an YM-30 spin filter (Amicon, Merck Millipore, Billerica, MA). Fragments greater than 100 bp were retained and eluted from the membrane, with ~ 90% recovery. Bar-coded paired-end SOLiD sequencing libraries were prepared using either SOLiD (Applied Biosystems/Life Technologies, Carlsbad, CA) or NEB (New England Biolab, Ipswich, MA) DNA library preparation kits. The cDNA was end repaired, and then barcoded SOLiD DNA sequencing adaptors were ligated to 1 microgram of input cDNA according to kit instructions. Ligated library product was size selected using a Pippin gel electrophoresis system (Sage Biosciences, Beverly, MA). The prepared library was enriched for correctly adapted product using 5-8 cycles of PCR.
identifiers.namespace: GEO
identifiers.id: GSM1675585