GSM4663044: Regorafenib, 135_13 (RNA-seq); Homo sapiens; RNA-Seq
Total RNA for analysis of mRNA was extracted from formalin-free alcoholic-based fixative FineFIX® and paraffin-embedded samples by MiRNeasy FFPE minikit (Qiagen, Venlo, Netherlands). RNA was quantified with Qubit 3.0 and Qubit RNA HS Assay kits (Applied Biosystems). RNAseq libraries were prepared from 100 ng of total RNA using the QuantSeq 3'mRNA-Seq Library Prep Kit-FWD (Lexogen, Vienna, Austria) according to manufacturer's instructions including the recommendation for FFPE samples. Briefly, after synthesizing the first strand using Oligo-dT and removing the RNA template, the second strand synthesis was initiated by random primer and DNA polymerase. During this step, Unique Molecular Identifiers (UMIs) were added to the libraries between the partial P5 adapter and the random priming sequence. After a magnetic beadbased  purification step, the libraries were then amplified by 16 PCR cycles and labelled with different single indexes. Quality and size of generated libraries were assessed using High sensitivity D1000 DNA assay Screen Tape (Agilent) and quantified by Qubit HS (Thermofisher). Libraries were then pooled at equimolar concentrations and sequenced on a NextSeq 500 sequencer (Illumina), by applying standard manufacturer protocols.
GEO Accession
GSM4663044