title: RNA-seq analysis of the budding yeast ctk1∆ and rad6∆ strains and prp45(1-169) ctk1∆ and prp45(1-169) rad6∆ double mutant strains
design: RNA-seq analysis of the budding yeast ctk1∆ and rad6∆ strains and prp45(1-169) ctk1∆ and prp45(1-169) rad6∆ double mutant strains
library_construction_protocol: Thirty milliliters of yeast cultures were grown at 30 °C in the YPAD medium (1% yeast extract, 2% peptone, 0.01% adenine, 2% glucose). Cells (8 ml of culture) were harvested by centrifugation (1000 g, 3 min, room temperature) at the exponentional growth phase (OD600 ~1.8). Cell pellets were stored at -80 °C. Thaw cell pellets on ice (~5 min). Add 400 ul of extraction buffer (1 mM EDTA, 0.1 M LiCl, 0.1 M Tris-HCl pH 7.5, 1% SDS), 400 ul of glass beads and 400 ul of acidic phenol (pH 4.3):chloroform:isoamylalcohol (25:24:1) solution. Disrupt the cells in FastPrep-24 (MP Biomedicals) (speed 6, duration 25 s) using 3 shaking cycles with 5 minutes incubations on ice in between. Pellet the debris by centrifugation (20 000 g, 10 min, 4 °C). Transfer the supernatant to a new microcentrifuge tube. Add 30 ul of 40% KAc (pH 5.5) and 400 ul of phenol (pH 4.3):chloroform:isoamylalcohol (25:24:1) solution. Mix vigorously by vortexing for 30 s and repeat the centrifugation step. Transfer the upper phase to a new microcentrifuge tube, add 1 ml of 96% EtOH, mix by inverting the tube and let RNA precipitate for at least 1 h at -20 °C. Pellet the RNA by centrifugation (20 000 g, 15 min, 4 °C). Remove the supernatant and rinse the pellet with 70% EtOH. Let the RNA pellet dry. Dissolve the RNA pellet in 150 ul of DEPC-treated water and measure RNA concentration on Nanodrop 2000 (Thermo Scientific). Take the volume corresponding to 150 ug of RNA. Add DEPC-treated water to the final volume of 175 ul, add 20 ul of DNase buffer and 5 ul of DNase from the MasterPure Yeast RNA purification kit (Epicentre). Finish the RNA preparation with this kit according to the manufacturer's protocol, starting with step 4 in section B of the protocol. After the last rinse with 70% EtOH (step 12), add 500 ul of 70% EtOH to the RNA pellet and store the samples at -20 °C. Strand-specific paired-end RNA-seq libraries were constructed from poly(A)-enriched RNA samples by BGI Genomics using their standard procedure.