title: GSM2723029: HGSC_JB5841; Homo sapiens; RNA-Seq
library_construction_protocol: DNA and RNA were co-isolated from frozen tissue tumor blocks, and RNA was isolated from normal tissues utilizing Ambion’s ToTALLY RNA™ RNA Isolation Kit (Part Number AM1910, ©Ambion, Inc.).  DNA concentration was confirmed using a PicoGreen protocol. RNA quality was confirmed by utilizing an Agilent Bioanalyzer and assigning an RNA Integrity Number (RIN) to each sample. RNA sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit.  Briefly, 500ng of total RNA was purified by Ribo-Zero to remove rRNA and fragmented by divalent cations under elevated temperature. The fragmented RNA underwent first strand synthesis using reverse transcriptase and random primers.  Second strand synthesis created the cDNA fragments using DNA polymerase I and RNaseH.   The cDNA fragments then went through end repair, adenylation of the 3’ ends, and ligation of adapters.  The cDNA library was enriched using 10 cycles of PCR and purified.