GSM3209234: hASTRO-control-1; Homo sapiens; RNA-Seq
Total RNA was extracted using Quiazol lysis reagent (Qiagen) and RNAeasy extraction kit (Qiagen) according to manufacturer's instructions. The RNASeq libraries were prepared from total RNA using the TruSeq®Stranded mRNA LT Sample Prep Kit (Illumina Inc., Rev.E, October 2013).  The mRNA was fragmented in the presence of divalent metal cations and at high temperature (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, this allowed to achieve the strand specificity. The blunt-ended double stranded cDNA was 3´adenylated and Illumina indexed adapters were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay.