GSM8828788: Surgical specimen for patient DNS21; Homo sapiens; RNA-Seq
RNA was extracted from O.C.T.™ samples at baseline and at the time of surgery. Before extraction, a section of the sample was stained with H&E to evaluate the percentage of tumor content. A tumor cellularity of at least 30% was deemed necessary for RNA extraction. Total RNA was isolated from tumor specimens using the Maxwell® RSC simplyRNA Tissue Kit (AS1340 Promega), following the manufacturer's instructions Total RNA samples (average 100 ng, range 0.2-300 ng) were processed into cDNA sequencing libraries with the "QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 015) *with a UMI Second Strand Synthesis module*. Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. While primers from both steps contain Illumina-compatible sequences, random primers additionally feature 6 nt long Unique Molecular Identifier (UMI) tags. Libraries are completed by PCR, and sequenced on an Illumina instrument by following manufacturer's protocols.