characteristics.text: 2025-02-01
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: Nuclear Dynamics, CNRS UMR 3664, Institut Curie, PSL Research University, Paris 75005, France
characteristics.characteristic: INSDC center name
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS20850101
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-14337:WT_DS_INPUT_1
characteristics.characteristic: Submitter Id
characteristics.text: 1
characteristics.characteristic: biological replicate
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: brewer's yeast
characteristics.characteristic: common name
characteristics.text: Protocols: For every timepoint in the kinetics of quiescence entry, 100 OD600 of cells were harvested for chromatin immunoprecipitation. For each of the two biological replicates, WT Rpb3 (yAT1684) and FLAG-tagged (yAT3239, yAT3555) strains were grown with the same batch of YPD and processed together for chromatin immunoprecipitation and imaging.  To recover only the highly dense population as cells progress into stationary phase in the nutrient exhaustion kinetics, all samples after diauxic shift (24h, 48h, Q6d) were crosslinked, then purified by gradient sorting, as described in Allen et al., 2006. 4.8x109 (400 OD600) cells were collected and re-suspended in sterile water to reach 1.2x107 cells/mL (1 OD600/mL) and immediately cross-linked with 1% paraformaldehyde (32% stock solution, Sigma) for 15 minutes, shaking, at room temperature, then quenched for 5 minutes with 2.5 M glycine (125 mM final). Then, crosslinked cells were pelleted at 4000 rpm for 20 minutes, washed and re-suspended in 50 mM Tris pH 7.5, 1X, before sorting on a density gradient (see above). The lower fraction, corresponding to highly dense (HD) cells, was collected, and washed twice with TBS 1X pH 7,6. 100OD600 were pelleted and flash-frozen in liquid nitrogen for storage at -80 °C before ChIP. To harvest Q-like cells for ChIP (quiescence by abrupt starvation), 400 OD600 of live cells were gradient-sorted after 24h of growth in YPD following the protocol described above. Then, 100 OD600 of highly dense cells were washed and re-suspended in H2O for 24h, before cross-linking and pelleting as above.   Two biological replicates were processed independently for chromatin immunoprecipitation, and all libraries for sequencing were prepared simultaneously. Unless indicated, all the following steps were carried out at 4°C. For every 20 OD600 of cells, 40 µL of protein G magnetic beads (New England Biolabs) were washed with wash and bind buffer(0.1M NaP, 0.01% Tween 20 pH 8.2), 7.5% BSA was added and incubated on a spinning wheel at 4°C for 3h. 4μL of FLAG antibody (Sigma-Aldrich, M8823) was added to every 4 µL of protein G magnetic beads, which were then incubated on a spinning wheel at 4°C for 4h.  Pellets were thawed and re-suspended in 500μL of IP buffer (20% SDS, 20% Triton X 100, 0,5M EDTA pH 8.0, 1M Tris pH 8.0, 5M NaCl, 7,5 % BSA, 10 mg/ml tRNA) with 2,5μL of Protease Inhibitor (Sigma-Aldrich P1816) and 2 OD600 nm equivalent of calibration cells (S. pombe) for every 100 OD600 nm of experimental cells. Using a FastPrep instrument (MP Biomedicals), exponential phase samples were mechanically lysed with 500 μm zirconium/silica beads (Biospec Products) by three cycles of 30 sec, intensity (6msec-1). All other samples were lysed with 710-1180 μm glass beads (SIGMA, G1142) by five cycles of 30 sec, intensity (6msec-1). Each bead beating cycle was followed by 5 min incubation on ice. Chromatin corresponding to 100 OD600nm was fragmented in 15 mL Bioruptor tubes (C30010017) to a minimum 200 bp and average size of 450 bp by sonication using Bioruptor Pico XL (Diagenode) for 30 min at high power with 30 sec on/30 sec off. Lysate was centrifuged 10 min at 4,500 rpm and 10 μL were saved as whole cell lysates (INPUT DNA). Antibody and protein G beads were recovered using a magnetic beads purification magnet (Invitrogen) and re-suspended in cleared lysate for incubation overnight on a spinning wheel. Magnetic beads were washed sequentially with lysis buffer, twice with RIPA buffer (0.1% SDS, 10 mM Tris at pH 7.6, 1 mM EDTA at pH 8, 0.1% sodium deoxycholate, and 1 % Triton X- 100), twice with RIPA buffer supplemented with 300 mM NaCl, twice in LiCl buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), with TE 0.2% Triton X-100 and with TE. Input was diluted 10X with elution buffer (50 mM Tris, 10 mM EDTA at pH 8, 1% SDS) and beads were resuspended in 200 μL elution buffer, then incubated, shaking at 800 rpm, for 20 minutes at 65ºC. Proteins were digested with proteinase K at 0,1 mg/mL. Crosslinking reversal was performed by heating samples overnight at 65°C. Samples were treated with RNase A for 1h at 37°C, and the remaining DNA was purified on QIAquick PCR purification columns. Libraries were prepared with TruSeq®ChIP Illumina kits. All libraries were amplified with 12 PCR cycles.
characteristics.characteristic: description
characteristics.text: Diauxic_shift
characteristics.characteristic: developmental stage
characteristics.text: WT_Rpb3-FLAG
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: 1
characteristics.characteristic: replicate
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: scientific_name
characteristics.text: 1
characteristics.characteristic: technical replicate
characteristics.text: WT_DS_INPUT_1
characteristics.characteristic: title