GSM9408889: NES052; Homo sapiens; RNA-Seq
Freshly frozen tissue specimens were homogenized in RLT buffer, and total RNA was extracted using the Qiagen RNeasy Mini Kit following the manufacturer's protocol. RNA was eluted in RNase-free water. RNA quantity and purity were assessed using NanoDrop spectrophotometry, and RNA integrity was evaluated with Agilent Bioanalyzer. RNA-seq libraries were prepared using the Illumina TruSeq RNA Exome (RNA Access) Library Preparation Kit according to the manufacturer's instructions. Briefly, 50–100 ng of total RNA was subjected to rRNA depletion, followed by first- and second-strand cDNA synthesis. The resulting cDNA fragments were end-repaired, A-tailed, and ligated to Illumina indexed adapters. Libraries were amplified by PCR and subjected to exome capture using the TruSeq RNA Exome probe set. Captured libraries were enriched, purified, quantified, and assessed for library size distribution prior to sequencing on an Illumina NovaSeq 6000.