GSM6697798: AEP1 (WT), Q-modified; 6.25µM dCTP; Schizosaccharomyces pombe; RNA-Seq
Total RNA was isolated from 50 OD of cells. Cells were harvested and after addition of 1 mL of phenol, glass beads and vigorous shaking for 5 min, samples were centrifuged at 20.000 g for 5 min to clear the cell debris. Equal volume of phenol/chloroform/isoamylalcohol was added to the aqueous phase and centrifuged at 20.000 g for 5 min. After mixing the upper phase with an equal volume of chloroform followed by centrifugation at 20.000 g for 5 min, the RNA was precipitated. Extraction of the RNA was performed as described in the "SAMPLEs" section. 500 ng RNA was hybridized to 500 nM of the respective stemloop-primer with the following thermocycling program: 95 °C for 2 min, 65 °C for 30 sec, ramp down to 4 °C with 5 °C/30 sec. After hybridization and incubation on ice for 1 min, 100 µM of each dNTP and 100 nM RT-KTq I614 DNA polymerase were added. The reverse transcription was carried out in reaction buffer (50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 25 mM MgCl2, 0.1% Tween 20) in a total volume of 20 µL. For experiments using decreased dCTP concentrations, 25 µM, 12.5 µM and 6.25 µM dCTP were added to the RT reaction. Following incubation at 55 °C for 1 h and incubation on ice for 10 min, the cDNA was amplified using Taq polymerase. Amplification was performed with 5 µL cDNA in reaction buffer containing 2.5 µM dNTPs, 2 mM MgCl2, 1 x Taq buffer+ KCl and 1.25 U Taq polymerase (Thermo Fisher) in a total volume of 200 µL. Primers binding at the 3' end contained a barcoded sequence for multiplexing the PCR products. Samples were separated on a 2% agarose gel, and the respective PCR products were gel-purified using the QIAquick Gel Extraktion Kit (Qiagen) according to the manufacturer's protocol. Library preparation of amplified tRNAs for deep sequencing was performed using the NEXTflexR qRNA-SeqTM Kit v2 – Set C (Bioo Scientific) according to the manufacturer's protocols.