GSM5239495: scrambled3; Homo sapiens; RNA-Seq
RNA was extracted with QIAZOL Lysis Reagent and RNeasy Lipid Tissue Mini Kit (QIAGEN) . RNA concentration and integrity were measured using a Nanovue Spectrophotometer (GE Healthcare) and the QIAxcel Advanced Instrument (QIAGEN), respectively. DNase I-treated total RNA (1 µg/reaction) was reverse transcribed with the QuantiTect Reverse Transcription (RT) kit (QIAGEN), using a mix of random hexamer and oligo-dT primers, as described. cDNAs were amplified in duplicate by real-time PCR using Maxima SYBR Green (Thermo Scientific) or GoTaq™ qPCR Master Mix (Promega), the LightCyclerTM480 (Roche) or Rotor-GeneTMQ (QIAGEN) instruments. Libraries were prepared with NEBNext Ultra II Directional Kit (New England Biolabs, Ipswich, MA, USA) In brief, 200 to 300 ng total RNA was used as input in the polyA enrichment module protocol. Enriched samples were fragmented and transcribed into cDNA. Following universal adapter ligation, samples were barcoded using dual indexing primers.