GSM5240602: CE_26; Homo sapiens; RNA-Seq
Total RNA was prepared from 5µm FFPE tissue sections of microdissected ITF using the Agencourt FormaPure kit (A33341; Beckman Coulter, Indianapolis, IN, USA) and following the manufacturer's instructions. RNA concentration was determined with Qubit 4 Fluorometer and Qubit® RNA HS Reagent (Thermo Fisher Scientific, Waltham, MA, USA). HTG EdgeSeq Chemistry was employed to synthetize the RNA-Seq library. Briefly, target capture was performed by hybridizing the mRNA with Nuclease Protection Probes (NPPs). The S1 nuclease was added to the mix, producing a stoichiometric amount of target mRNA/NPP duplexes. This reaction was blocked by enzyme heat denaturation of S1. The samples were randomized before inclusion in the HTG EdgeSeq system to reduce potential biases in the run. Each hybridized sample was used as template to set up PCR reactions with specially designed tags, sharing common sequences that are complementary to both 5'-end and 3'-sequences of the probes, and common adaptors required for cluster generation on an Illumina sequencing platform. In addition, each tag contains a unique barcode used for sample identification and multiplexing. After PCR amplification, a clean-up procedure was performed using Agencourt AMPure XP (Beckman Coulter). The library was quantified by quantitative PCR, using KAPA Library Quantification (Roche), according to the manufacturer's instructions. All samples and controls were quantified in triplicate and no template control was included in any run.