GSM5031980: C40; Homo sapiens; RNA-Seq
Total RNA was extracted from three 30μm scrolls cut from FFPE tissue blocks containing 90% tumor or more, using the RecoverAll total nucleic acid isolation kit (Ambion/Thermo Fisher Scientific, Waltham, MA) per the manufacturer's instructions. RNA was then passed through concentration and clean-up columns (Norgen Biotek, Thorold, ON) and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Capture-based RNAseq was performed using a modified SureSelect strand-specific RNA library prep and target enrichment protocol (Agilent Technologies, Santa Clara, CA). Briefly, 200ng of FFPE-derived total RNA was heated to 65 °C for 5 min and used for first-strand cDNA synthesis. Second-strand cDNA was synthesized, and the ends repaired. The 3ʹ ends of ds-cDNA were adenylated, ligated with adapters and fragments were PCR-amplified. PCR product (700ng) was hybridized overnight to SureSelect XT Human All Exon V5+lncRNA baits. The hybridized fragments were captured on magnetic beads and PCR-amplified using uniquely barcoded index primers. RNAseq. Barcoded libraries were pooled and underwent paired-end 150bp sequencing on an Illumina HiSeq4000 instrument (Illumina, San Diego, CA).
GEO
GSM5031980