GSM6049470: BY-batch3-1st-3h; Saccharomyces cerevisiae; RNA-Seq
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. RNA-Seq: First rRNA was depleted  from total RNA with Illumina ribozero Gold kit. Then stranded RNA library was generated with NEBnext Ultra Directional RNA Library Prep kit for illumina.  MNase and ChIPSeq-Seq:  100 ml culture per S. cerevisiae sample and 30 ml of S. pombe culture was crosslinked using 1% formaldehyde for 15 minutes at room temperature. Formaldehyde was quenched by 0.125 M glycine for 5 minutes. Then cells were washed three times with cold TBS, flash-frozen in liquid nitrogen and stored at -80 °C.  Frozen cell pellets were resuspended in zymolyase solution (1 M sorbitol, 50 mM Tris-HCl pH 7.5, 1% beta-mercaptoethanol, 0.1 U/µl zymolyase). The zymolyase digestion proceeded at +37 °C for 30 min (S. cerevisiae) or 90 min (S. pombe). Spheroplasts were isolated by centrifugation at 6000xg for 10 minutes at +4 °C. Spheroplasts were resuspended in NP buffer (10 mM Tris-HCl pH 7.5, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40 (Tergitol), 1% beta-mercaptoethanol, 0.5 mM spermidine, 1% yeast protease inhibitor cocktail). The suspensions were pre-warmed and then 0.5 U/µl of MNase for S. cerevisiae and 2 U/µl for S. pombe was added per sample. MNase digestion proceeded at 37 °C for 40 min (S. cerevisiae) or 30 min (S. pombe) and stopped by addition of EGTA. The supernatants containing chromatin fragments were collected and  diluted with 1 ml RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate) with 1% yeast protease inhibitor cocktail. Equal volumes of S. pombe chromatin were spiked in to S. cerevisiae chromatin samples at this point, corresponding to around 3% of S. cerevisiae DNA.  Each sample was immunoprecipitated with anti-H3K4me3 (Abcam ab8580) and anti-H3K4me2 (Abcam ab7766) antibodies. Protein A/G beads (Pierce) were coupled to antibodies and resuspended in diluted chromatin and rotated o/n at 4 °C.  The next day, beads were washed with RIPA buffer, RIPA-500 buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% v/v NP-40, 0.5% w/v sodium deoxycholate), and TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8). Volume of each wash was 150 µl. Chromatin was eluted from beads in 2x10 µl ChIP elution buffer (Tris-HCl pH 8 50 mM, 1% SDS, 10 mM EDTA pH 8). The immunoprecipitated chromatin as well as a 20 µl sample of input chromatin were decrosslinked by TE, RNase cocktail, Proteinase K and 6 µl SDS (10% w/v), incubating overnight at 65 °C. DNA was purified by ethanol precipitation. Illumina sequencing libraries were prepared from the DNA using the NEBNext Ultra II kit without dual size selection and using 1.4X volume of AMPure XP beads for the purification steps. The libraries were sequenced on Illumina's NextSeq 500, paired-end, 39 bases from each end     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 and 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.