title: GSM1147075: Mutant Staufen associated mRNA; Homo sapiens; RNA-Seq
library_construction_protocol: TAP-associated RNAs were obtained by treatment of Calmodulin-agarose resin with 0.2 mg/ml Proteinase K and 0.5% SDS in TNE for 1 hour at 37C. After phenol extraction, RNAs were precipitated with 2 ethanol volumes and 20 μg of glycogen (Roche) and resuspended in DEPC-treated H2O. Before cDNA synthesis, each RNA preparation was monitored using the Agilent 2100 Bioanalyzer (Agilent technologies). RNA samples of hStau1-TAP (treated or not with RNase T1) and the untreated hStau1-Mut (100 ng each) were used to generate cDNA libraries according to Illumina mRNA-seq protocol at Fasteris SA Company (Plan-Les-Ouates, Switzerland) but the poly (A+) RNA purification step was skipped. Furthermore, mRNA fragmentation was omitted in the T1-RNase treated hStau1-associated RNA, as it already contained RNAs about 100 nt in size. cDNA libraries were subjected to ends repair reaction and Illumina adapter ligation and further purified from polyacrilamide gels. This purification step excluded the identification of putative small RNAs present in the samples.  cDNA libraries were sequenced at Fasteris SA in an Illumina HiSeq2000 sequencer. Reads length were 100nt in a single-read run cycle.