GSM7745499: cancer cells from patient 2 after chemotherapy; Homo sapiens; RNA-Seq
Total RNA was isolated from primary cultures of cancer cells using RNeasy Mini Kit (Qiagen). DNase treatment was carried out with TURBO DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA), in volumes of 50 µl. RNA cleanup was performed with the Agencourt RNA Clean XP kit (Beckman Coulter). The concentration and quality of the total RNA were checked by the Quant-it RiboGreen RNA assay (ThermoFisher) and the RNA 6000 Pico chip (Agilent Technologies), respectively. Enrichment of polyadenylated RNA and library preparation was performed with NEBNext Ultra II Directional RNA Library Prep Kit (NEB), according to the manufacturer's protocol. The library underwent a final cleanup using the Agencourt AMPure XP system (Beckman Coulter) after which the libraries' size distribution and quality were assessed using a high sensitivity DNA chip (Agilent Technologies). Libraries were subsequently quantified by Quant-iT DNA Assay Kit, High Sensitivity (ThermoFisher). Finally, equimolar quantities of all libraries (12 pM) were sequenced by a high throughput run on the Illumina HiSeq 2500 using 2 × 100 bp paired-end reads and a 5% Phix spike-in control. RNA libraries were prepared for sequencing using standard Illumina protocols