GSM3219610: ICM49; Homo sapiens; RNA-Seq
Frozen tissue was placed in liquid nitrogen and broken up using mortar and pestle to obtain a piece approximately 2 x 2 x 2 mm in size, macroscopically free of fatty infiltration, fibrosis, and blood. Tissue was placed immediately in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) and mechanically homogenized using an IKA T25 Ultra-Turaxx homogenizer. Tissue was homogenized for approximately 60 seconds, or until no visible chunks of tissue remained. RNA was extracted using the mirVana miRNA isolation kit (Thermo Fisher Scientific) enriched for total RNA isolation according to manufacturer's instructions with the exception of replacing the Lysis/Binding Buffer with TRIzol. All samples were DNase treated using TURBO DNA-free Kit (Thermo Fisher Scientific). RNA was quantified at 260nm using a NanoDrop1000 (Thermo Fisher Scientific), and RNA integrity (RIN) was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Assay (Agilent, Santa Clara, CA). All samples were required to demonstrate RIN ≥ 7.0, and ranged from 7.0-9.3. PolyA transcripts were isolated from 1µg total RNA using oligo-dT beads and the cDNA libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit and protocol from Illumina (Illumina Inc., San Diego, CA).
GEO Accession
GSM3219610