GSM6744266: Slamseq_by_Pulse_Chase_P_t10_r3; Saccharomyces cerevisiae; OTHER
RNA was extracted with MasterPure RNA extraction kit from Lucigene  The DNA was purified with phenol, precipitated with ethanol and sodium acetate at -20 °C, washed with cold 70% ethanol, and resuspended in 30 µl TE buffer. SLAMSeq: Metabolic labeling of newly synthesized RNA molecules was done as previously described (Voichek et al., 2016). Briefly, 4-thiouracil (4tU) (Sigma) was dissolved in NaOH(83mM), Newly synthesized RNA was labeled for indicated  time  spans  (10  min)  at  a  final  concentration  of  5mM  4-thiouridine  (4sU,  Carbosynth). MES buffer with a final concentration 10mM PH 5.9 was added to media to avoid pH change as a result of NaOH addition. At each time points before harvesting, 4tU was added to media for ten minutes ahead of desired time point.  cells were snap frozen in liquid nitrogen immediately at the time point required.     Pulse and Chase: briefly, 4-thiouracil (4tU) (Sigma) was prepared as explained above. Prewarmed 4tU was added to culture media (YPD with MES buffer) containing wild-type cells. After 1 hour labelling with 4tU, cells were washed and resuspend in YPGal media and time points were collected at 0, 10 and 30 minutes (t0, t10, t30, t0', t10', t30') by centrifugation and snap frozen in liquid nitrogen immediately. Between the first and second exposure to YPGal, cells were cultured for 3 hours in YPD.     RNA purification was done with MasterPure Yeast RNA Purification Kit. Total RNA was subjected to thiol(SH)-linked alkylation by iodoacetamide (Sigma, 10 mM) at 50C for 15 minutes (Herzog et al., 2017), the reaction was stopped by 20 mM DTT and RNA  was  re-purified by ethanol precipitation. After rRNA depletion with RiboPool Depletion Kit, library was prepared by Ultra™ II Directional RNA Library Prep Kit for Illumina® and sequenced single end with read length 150bp on Nextseq 500 Illumina sequencer.