title: GSM8895145: ccRCC, pretreatment tumor, TKI/IO treatment group, sample 39; Homo sapiens; RNA-Seq
library_construction_protocol: For snap frozen tissues, 20-30mg frozen tissue was homogenized in 1mL TRIzol Reagent (ThermoFisher catalog # 15596018) and phase separation was induced with 200 µL chloroform. RNA was extracted from the aqueous phase using the miRNeasy Micro or Mini Kit (Qiagen catalog # 217084/217004) on the QIAcube Connect (Qiagen) according to the manufacturer's protocol with 350 µL input. Samples were eluted in 15-16 µL RNase-free water. For FFPE tissues, sections were deparaffinized using the mineral oil method. Briefly, 800µL mineral oil was mixed with the sections and the sample was incubated at 65°C for 10 minutes. Phases were separated by centrifugation in 360µL Buffer PKD and Proteinase K was added for digestion. After a three-step incubation (65°C for 45', 80°C for 15', 65°C for 30') and additional centrifugation, the aqueous phase containing RNA was removed and DNase treated. The RNA was then extracted using the RNeasy FFPE Kit (QIAGEN catalog # 73504) according to the manufacturer's instructions. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 432ng-1µg of total RNA with DV200 values from 24-83% underwent ribosomal depletion and library preparation using the TruSeq Stranded Total RNA LT Kit (Illumina catalog # RS-122-1202) according to instructions provided by the manufacturer with 8 cycles of PCR.