GSM4998273: 4722-RNAseq; Homo sapiens; RNA-Seq
Prior to analysis, frozen tumor block was assessed by a board-certified pathologist (SQ) to assess for adequate tumor content. Total RNA was purified from tumor specimens using the RNeasy Plus Micro Kit (Qiagen) with genomic DNA removal. RNA integrity (RIN) was determined by electrophoretic tracing using an Agilent Bioanalyzer. RNAseq libraries were constructed from samples (RIN >7.0) using the Illumina TruSeq Stranded Total RNA kit with Ribo-Zero rRNA depletion. Indexed libraries were sequenced on an Illumina NextSeq 500 DNA sequencer using 150x150-nt paired end reads, generating >40 million reads per sample (12 samples per flow cell) with >80% of sequences achieving >Q30 Phred quality scores. RNA-Seq analysis was performed following the standard pipeline established by The Cancer Genome Atlas (TCGA) and the National Cancer Institute Genomic Data Commons (GDC). Briefly, quality of raw sequencing reads was assessed by FASTQC analysis (Babraham Bioinformatics). Sequence reads were aligned using the Spliced Transcripts Alignment to a Reference (STAR) sequence aligner with a two-pass approach and gene counts determined using HTSeq software. The RNA-Seq data was reported as raw counts, FPKM (Fragments Per Kilobase of transcript per Million mapped reads), and FPKM-UQ (Upper 75% Quantile). RNA libraries were prepared for sequencing using standard Illumina protocols