GSM2359148: Tumor CDC3; Homo sapiens; RNA-Seq
Frozen patient tumor samples and matched normal kidney tissues were obtained from the tissue bank at the participating institutions.  The sequencing libraries were prepared from 1µg total RNA using a TruSeq Stranded Total RNA kit (Illumina Inc) according to the manufacturer’s instructions. After rRNA depletion from total RNA, the remaining RNA was purified, fragmented, and primed for cDNA synthesis.  Fragmented RNA was reverse transcribed into first strand cDNA using random primers.  The RNA template was then removed and a replacement strand synthesized, incorporating dUTP in place of dTTP to generate ds cDNA.  AMPure XP beads were used to separate the ds cDNA from the second-strand reaction mix resulting in blunt-ended cDNA.  A single ‘A’ nucleotide was then added to the 3’ ends of the blunt fragments.  Multiple indexing adapters, containing a single ‘T’ nucleotide on the 3’ end of the adapter, were ligated to the ends of the ds cDNA, preparing them for hybridization onto a flow cell.   Libraries were purified and validated for appropriate size on a 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Inc.).   The DNA library was quantitated using a KAPA Biosystems qPCR kit, and then pooled in an equimolar fashion to a final concentration of 2nM.  Each pool was denatured, diluted to 10pM, and clustered to individual lanes of a HiSeq Flow Cell using an Illumina cBot and the corresponding paired-end TruSeq V3 cluster kit.
GEO Accession
GSM2359148