title: GSM3104652: s_5_16_NDLl3_Fr; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated from frozen samples using the miRNeasy FFPE Kit (Qiagen) according to the manufacturer's protocols.  Total RNA was eluted from the columns in nuclease-free water and stored at -80°C. Total RNA-Seq sequencing libraries were prepared from both intact RNA derived from flash-frozen samples and severely fragmented RNA isolated from FFPE samples.  Briefly, rRNA-depleted RNA was prepared from total RNA input (1-5 ug) using the Ribo-Zero rRNA Removal Kit (Epicentre), which selectively depletes rRNA by hybridization to biotinylated capture probes followed by microsphere bead capture. Subsequently, directional Total RNA-Seq libraries were prepared from rRNA-depleted RNA (50 ng) using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) according to the manufacturer's protocol. Briefly, 5',3'-di-tagged cDNA was synthesized using tagged random hexamer-primed cDNA synthesis primers followed by annealing of 3'-terminal-tagging oligo (TTO) and extension with DNA Polymerase. The cDNA was then purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.) or with the MinElute PCR Purification Kit (Qiagen), depending upon the source of RNA from flash-frozen or FFPE samples respectively. Illumina adaptor sequences and indexes were then incorporated during library amplification with the appropriate PCR primers and high-fidelity FailSafe PCR Enzyme Mix. Subsequently, the libraries were purified in a similar manner to that described above for the cDNA preparations. The ScriptSeq v2 libraries were then quantitated with the Qubit fluorometer (Invitrogen Life Sciences) and insert sizes determined with the Agilent 2100 Bioanalyzer. The molar concentration of PCR-competent sequencing templates in the libraries was then determined using by quantitative PCR with the KAPA Library Quantification Kit (Kapa Biosystems, Inc.).