GSM8085069: TLE-HS_36; Homo sapiens; RNA-Seq
For RNA isolation, human tissue was homogenized in 700 µl Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). Total RNA including the microRNA (miRNA) fraction was isolated using the miRNeasy Mini Kit (Qiagen Benelux, Venlo, The Netherlands) according to the manufacturer's instructions. The concentration and purity of RNA was determined at 260/280 nm using a Nanodrop spectrophotometer (Ocean Optics, Dunedin, FL, USA) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA, USA). Only samples with RNA integrity number (RIN) equal or greater than 6.0 were used for sequencing. All library preparation and sequencing were performed by GenomeScan (Leiden, The Netherlands). The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) was used to process the samples. Sample preparation was performed according to the protocol NEBNext Ultra II Directional RNA Library prep Kit for Illumina (NEB #E7760S/L). Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads. After fragmentation of mRNA, cDNA synthesis was performed. Next, sequencing adapters were ligated to the cDNA fragments followed by polymerase chain reaction amplification. Clustering and DNA-sequencing was performed using the NovaSeq6000 (Illumina, Foster City, CA, USA) in accordance with manufacturers' guidelines. All samples underwent paired-end sequencing of 150 nucleotides in length; the mean read depth per sample was 47 million reads.