GSM4726765: Sample_S18; Homo sapiens; RNA-Seq
10 μm sections were cut from the FFPE blocks and collected in 2 ml microcentrifuge tubes. The number of sections per sample was dependent on the size of the tissue; 4 sections for small sized samples, 3 - 4 sections for medium sized samples, 2 - 3 sections for large sized samples and 1 – 2 sections for very large sized samples. RNA extraction and purification were performed using the QIAGEN RNeasy FFPE kit following the manufacturer's protocol. Whole transcriptome sequencing was performed using Illumina's Next Generation Sequencing RNA-Seq platform following established protocols. RNA samples were assessed for quantity and integrity using the NanoDrop 8000 spectrophotometer V2.0 and Agilent 2100 Bioanalyser. From each sample, 100 ng of total RNA was used to prepare RNA libraries using the KAPA Stranded RNASeq Kit with RiboErase. Prior to first strand cDNA synthesis, fragmentation was carried out using incubation conditions recommended by the manufacturer for degraded samples (65°C for 1 minute), and 14 cycles of PCR were performed for final library amplification. The libraries produced were quantified using the Qubit 2.0 spectrophotometer and assessment of the average fragment size was performed using the Agilent 2200 TapeStation. The Illumina NextSeq®500 was used to generate 75 bp paired-end reads for each library.