GSM1166027: ABRF-454-CNL-A-2; Homo sapiens; RNA-Seq
NA Poly A; Library synthesis and sequencing was performed with MAQC A and MAQC B samples at three core laboratory sites.  Each site constructed one cDNA library from each of the polyA-enriched RNA samples.  Enriched RNA (200 ng) was reduced to 19 µl in a vacuum centrifuge at 60 oC, followed by library construction using  the Roche cDNA Rapid Library Preparation Method Manual XL+ (May 2011) with the following modifications: (1) RNA Fragmentation Reagent kit (AM#8740, Life Technologies) was used in place of the RNA Fragmentation Solution; (2) all magnetic particle concentrator (MPC) pelleting steps were held for 2 minutes; (3) Roche rapid library multiplex identifier (RL-MIDs) adaptors were used for the adaptor ligation step; (4) the final libraries were quantified using a Qubit fluorometer (Life Technologies) and average fragment sizes were determined by analyzing 1 µl of the libraries on the Agilent Bioanalyzer 2100 using a High-Sensitivity DNA LabChip; and (5) the library concentrations were determined using the average fragment size from the Bioanalyzer analysis.  Final samples were diluted to 1x108 molecules/µl in Tris-HCl pH 8 buffer with 0.001% Tween-20.