GSM8729628: dhh1delta_2_csm_t30; Saccharomyces cerevisiae; OTHER
Metabolic labelling of newly synthesized RNA molecules was performed as previously described [PMID: 28945705, PMID: 36838506].  Briefly, 4tU (Sigma)  was dissolved in NaOH (83 mM) used for labelling RNA molecules. The final concentration of 4tU for YPD and CSM is 5mM (PMID: 26941319) and 0.2 mM (PMID: 30192227), respectively. MES buffer (pH 5.9) with a final concentration 10 mM was prepared with media to avoid the PH change due to 4tU addition. To perform pulse and chase experiment, RNA molecules were labelled with 4tU (prepared as above) for 1h during cell grown at mid exponential phase. Cells were washed and resuspend in prewarmed medium (YPD with MES buffer or CSM with MES buffer) without  4tU and time points were collected at 0, 15 and 30 mins by centrifugation and snap frozen in liquid nitrogen immediately. To perform thiol(SH)-linked alkylation, the reaction including 5 μg of RNA  was incubated with iodoacetamide (final concentration at 0.5 M) at 50 oC for 15 minutes [PMID: 28945705]. The reaction was stopped by adding 0.1 M DTT (final concentration at 20 mM). RNA was then precipitated by 3 M of sodium acetate and pure ethanol.  Purified RNA was subjected for ribosomal RNA depletion using RiboPools Depletion Kit (siTOOLs Biotech). Libraries were prepared by  Ultra™ II Directional RNA Library Prep Kit for Illumina® following manufacture instructions.  Sequencing was performed on Illumina Nextseq 2000 sequencer with single end read length for 150 cycles.