GSM4862232: PitNET11; Homo sapiens; RNA-Seq
Twenty to 30 mg of PitNET tissues was homogenised using a FastPrep-24™ homogenizer in RLT Plus buffer in Lysing Matrix D 1.5 ml tubes (MP Biomedicals, USA), and total RNA was extracted with AllPrep DNA/RNA Mini Kit (Qiagen, Germany) and stored at -80 °C. Total RNA quality control was carried out using Agilent 2100 bioanalyzer and an Agilent RNA 6000 Pico Kit (Agilent Technologies, USA) and concentration was measured using Qubit 2.0 fluorometer and Qubit™ RNA HS Assay Kit (Thermo Scientific, USA). Transcriptome libraries were prepared with the Low Input RiboMinus™ Eukaryote System v2 and Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA) following the manufacturer's instructions. Input amount for the transcriptome library preparation was 500 ng. The preparation of transcriptome libraries was carried out in relation to 1:1 based on patient samples, who had, or had no medical therapy before PitNET resection, to reduce any batch effects. Libraries were sequenced using the Ion Proton™ System for Next-Generation Sequencing (Ion OneTouch and Ion PI™ Hi-Q™ OT2 Reagents 200 Kit for emulsion PCR and Ion PI™ Hi-Q™ Sequencing 200 Solutions kit and Ion PI™ Chip V3 chips for sequencing, all from Thermo Fisher Scientific, USA). Libraries were sequenced 3 - 4 times to acquire at least 20 M reads per sample and at least 10 M would represent uniquely mapped reads, so that the rRNA content would not reach 50 % of the sample.