RNA-Seq of homo sapiens;  endometrial adenosquamous carcinoma cell-line (RL95-2; CRL-1671)
Total RNA was extracted from the endometrial cell line by TRIzol reagent and ethanol precipitation (TRIzol reagent; Invitrogen). To increase the efficiency of RNA extraction, 2 l glycogen (UltraPure Glycogen, Cat. no. 10814-010, ThermoFisher Scientific, Bleiswijk, Netherlands) was added to the lysis buffer per sample. The RNA pellet was washed three times by 70% ethanol. Quality and quantity of the extracted RNA samples were analyzed by Bioanalyzer Automated Electrophoresis instrument (Agilent technologies, Santa Clara, CA) using Agilent RNA 6000 Pico Kit (Agilent technologies) and Agilent Small RNA kit (Agilent technologies). Extracted RNA was affinity precipitated according to the manufacturers instruction of Click-iT Nascent RNA capture kit (Thermo Fisher Scientific, Waltham, MA; Cat. No. C10365). Ovation RNA-Seq System V2(NuGEN technologies, San Carlos, CA, Cat.No.7102-32) was used for cDNA library synthesis. The manufacturer's protocol was slightly modified to allow single strand cDNA to be synthesised (ssDNA) from on-bead RNA fragments. The modifications were as follows, 2 l of First Strand Primer Mix was added to 14 l on-bead RNA fragments and incubated for 5 min at 65C, followed by cooling on ice for 5 min. Then, 0.5 l of first strand enzyme mix and 5 l of first strand buffer mix were added to the mixture resulting in a final volume of 20 l. The mixture was incubated at 43.5 C for 60 minutes on an eppendorf thermomixer (700-800 rpm) to prevent the beads from settling. Finally, the mixture was thermalshocked at 85C for 10min and beads were rapidly immobilized by a magnet allowing the collection of cDNA from the supernatant. Ten l of first strand cDNA was used in the double strand synthesis step. Double strand cDNA synthesis was performed according to NuGEN manufacturers instructions. cDNA quality was measured by High Sensitivity DNA 1000 Assay Kit (Agilent technologies). Double stranded cDNA was subsequently used for barcoded library preparation. Libraries were prepared using the AB Library Builder System (Thermo fisher, Cat.No. 4477598) and Ion Xpress Plus Fragment Library Kit (Thermo fisher), according to the manufacturers instructions. The barcoded libraries were sequenced on two Ion 540 Chips (Thermo Fisher Scientific Inc, CA, USA, Cat. No. A27766) with four libraries per chip using the Ion S5 XL sequencer (Thermo Fisher Scientific Inc).