Leucine 100 mg / l 1
Saccharomyces cerevisiae
Protocols: For the experiments the cells were first grown over night in minimal media (14.4g / l KH2PO4, 0.5g /l MgSO4, 7.5g /l (NH4)2SO4, 1 ml /l tracemetal stock solution, 1 ml / l vitamin stock solution. The pH of the media was adjusted to 6.2-6.3 using KOH pellets. Trace metal stock solution components (per liter of stock solution): 15.0 g EDTA-Na2, 4.5 g CaCl2·2H2O, 4.5 g ZnSO4·7H2O, 3 g FeSO4·7H2O, 1g H3BO3, 0.84 g MnCl2·2H2O, 0.4 g Na2MoO4·2H2O, 0.3 g CuSO4·5H2O, 0.3 g CoCl2·6H2O and 0.1 g KI. Vitamin stock solution components (per liter of stock solution): 25 g myo-inositol, 1 g nicotinic acid, 1 g calcium pantothenate, 1 g pyridoxine HCl, 1 g thiamine HCl, 0.2 g 4-aminobenzoic acid and 0.05 g biotin.) with 2% glucose in shake flasks at 30°C and 200 rpm shaking. The next day 10 shake flasks were set up at a starting OD600 of 0.05 in 20 ml of minimal media with 2 glucose feed beads (Kuhner Shaker, 12mm high release glucose FeedBeads, Art. No. SMFB08001) at 30°Celsius and 200 rpm shaking. After 70 hours the cells were treated in duplicates with one of the following: no leucine, low level of leucine (final concentration 20 mg / ml), a high level of leucine (final concentration 100 mg / ml), a high level of isoleucine (final concentration 100 mg / ml) or a high level of valine (final concentration 100 mg /ml). After another 2 hours (72 hours of growth in total), 50 OD600 of cells were collected for Chromatin Immunoprecipitation followed by sequencing (ChIP-seq). DNA-protein complexes were cross-linked for 15 minutes at room temperature using a 1% final concentration of formaldehyde and the cross-liking reaction was then stopped by adding glycine for a final concentration of 125mM for 5 minutes at temperature. Finally, the cells were washed twice with TBS and then snap frozen in liquid nitrogen. The cells were thawed in ice-cold lysis buffer (50mM HEPES/KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Trition X-100, 0.1% Na-deoxycholate, 0.05% SDS and 0.2% Sigma P8215 protease inhibitor cocktail) and transferred to MP Biomedicals Lysing Matrix C glass bead tubes for disruption. For that the Precellys FastPrep was used with 6 cycles of 25 seconds and 7400 rpm. The cells were then transferred into a fresh 5ml tube and more lysis buffer was added to a final volume of 3 ml. Using a Branson digital sonifer 250 with a 1/8 inch microtip the cells were then sonicated for 5 minutes with pulses of 10 seconds on and 20 seconds off at an amplitude setting of 30%. After sonication the solution was centrifuged at 21.000 g for 15 min at 4°C to remove the cell debris. The immunoprecipitation was done using 15 µl of Pierce Anti-c-Myc magnetic beads over night at 4°C. After that the beads were washed with lysis buffer with out protease inhibitor and SDS, then with IP wash buffer (10 mM Tris-HCl pH 7.5, 250 mM LiCl, 2mM EDTA, 0.5% Triton X-100 and 0.5% Na-deoxycholate) and then with 10 mM Tris-HCl pH 8.0 plus 0.025% Tween20. After the washing, the DNA was eluted from the beads and the cross linking was reversed using Myc-elution buffer (25 mM Tris-HCl pH 8.0, 2 mM EDTA, 200 mM NaCl, 0.5% SDS) with 0.66 µg / µl protease K and 0.083 µg / µl RNase A. The DNA was extracted using the Promega ProNex size-selective purification system, using a double selection with first 1 x volume for an upper length cutoff of 1000bp and then 2 x volume for a lower cutoff of 100 bp. For sequencing the Oxford Nanopore PCR Barcoding Kit (SQK-PBK004) was used. The purified and size selected DNA were first prepared for adapter ligation using the NEBNext Ultra II End Repair / dA Tailing Module and cleaned up again using ProNex. The ligation of the Barcode Adapters (BCA) was done using the NEB Blunt/TA Ligase MasterMix for 15 min at 25°C. After clean-up using ProNex, the DNA was amplified with the Barcode Primers (LBW 01-10) for 25 cycles using Phusion DNA polyermase. The assigned barcodes for each sample are shown in Table 1.After a final clean-up using 1.75 x volume for a lower cutoff of 200bp the 10 samples were pooled in equal ratios to a total of 100 ng of DNA in 10µl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. Then the Rapid Adapters (RAP) were added and the Oxford Nanopore flow cell (version R9.4) was primed and loaded.Here the libraries were sequenced using the Oxford Nanopore MinKNOW software suite using two flow cells after each other to obtain a total of 4.5 million reads.
ENA-FIRST-PUBLIC
2020-07-01T04:04:27Z
ENA-LAST-UPDATE
2020-02-26T11:16:58Z
External Id
SAMEA6591076
INSDC center name
Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, SE-41296, Sweden
INSDC first public
2020-07-01T04:04:27Z
INSDC last update
2020-02-26T11:16:58Z
INSDC status
public
Submitter Id
E-MTAB-8820:Leucine 100 mg / l 1
broker name
ArrayExpress
common name
baker's yeast
growth condition
100 mg / l Leucine
sample name
E-MTAB-8820:Leucine 100 mg / l 1
scientific_name
Saccharomyces cerevisiae
strain
CEN.PK113-5D (URA- of CEN.PK113-7D)