GSM8605357: organoid, individual 33; Homo sapiens; RNA-Seq
Total RNA from organoids was extracted using the Monarch Total RNA Miniprep Kit (NEB, cat.: T2010S) following the manufacturer's protocol. RNA from parental tumors was extracted from FFPE tissue using the RNEasy FFPE Kit (Qiagen, cat. 73504), also according to the manufacturer's guidelines. In brief, tumor regions on slides were identified and marked by a board-certified pathologist, then scraped off the slides. Tissue from multiple sections, with a combined thickness of up to 40µm, was pooled before initiating RNA isolation as per the manufacturer's instructions. The amount of total RNA was quantified using the Qubit 2.0 Fluorometric Quantitation system (Thermo Fisher Scientific, Waltham, MA, USA) and the RNA integrity number (RIN) was determined using the 2100 Bioanalyzer instrument (Agilent, Santa Clara, CA, USA). RNA-seq libraries were prepared with the Watchmaker RNA Library prep kit with Polaris Depletion (Watchmaker Genomics, Inc., Boulder, CO, USA). All formalin-fixed, paraffin embedded (FFPE) samples, underwent special treatment adhering to the vendor's protocol. NGS library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA) and the size distribution was assessed using the 2100 Bioanalyzer instrument (Agilent, Santa Clara, CA, USA). For sequencing, samples were diluted and pooled into multiplex NGS libraries in equimolar amounts.