characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-07-22T17:01:21Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T20:02:25Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2627179
characteristics.characteristic: External Id
characteristics.text: EBI
characteristics.characteristic: INSDC center name
characteristics.text: 2014-07-22T17:01:21Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T20:02:25Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS497078
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2717:P16V H3K4me3 levels
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: baker's yeast
characteristics.characteristic: common name
characteristics.text: Protocols: Yeast cells were grown at 30 degrees C, shaking at 200 rpm to exponential phase in YPD medium (1 % yeast extract (Difco), 1 % bactopeptone supplemented with 2 % glucose). Cells were grown overnight in 5 ml cultures before dilution to 0.2 OD600 (0.5 x 107 cells/ml) in the appropriate volume of fresh media. Cells were harvested during exponential growth at 0.5 OD600 (1.25 x 107 cells/ml) by centrifugation (3000 rpm, 5 min). Cells grown to OD600 0.5 in 50 ml of appropriate media were fixed with 1 % formaldehyde in 45 ml PBS for 30 min at 22 degrees C followed by addition of glycine to 125 mM for 5 min. Cell pellets were collected by centrifugation (3000 rpm, 5 min) before washing twice with 10 ml cold PBS. Cells were resuspended in 500 ul cold FA-150 buffer (10 mM HEPES pH 7.9, 150 mM NaCl, 0.1 % SDS, 0.1 % sodium deoxycholate, 1 % Triton X-100) and broken using 1 ml glass beads on a MagnaLyser (Roche) at 4 degrees C. Sample volume was increased to 2 ml with FA-150 buffer before shearing of the fixed chromatin by sonication using a biorupter (Diagenode, 30 min, 1 min on, 20s off, medium setting). Chromatin was cleared by centrifugation (10 000 rpm, 15 min, 4 degrees C) and incubated with antibody (H3 Millipore/07-690 1:40 dilution, H3K4me3 Millipore/05-745R 1:40 dilution) in 1.5 ml siliconised Eppendorf tubes for 15-20 h rotating at 4 degrees C. Bound chromatin was immunoprecipitated for 90 min at 22 degrees C with 50 ul protein A-Sepharose pre-blocked with sonicated salmon sperm DNA. Beads and attached chromatin were pelleted by centrifugation (2600 rpm, 1 min) and washed with TSE-150 buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100) for 3 min, TSE-500 buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100) for 3 min, LiCl buffer (0.25 M LiCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1 % dioxycholate, 1 % NP-40) for 15 min and twice with TE, all at 22 degrees C. After washing, chromatin was eluted from the beads for 30 min at 65 degrees C with elution buffer (0.1 M NaHCO3, 1 % SDS). Addition of 350 mM NaCl and incubation for 3 h at 65 degrees C reversed the cross-links before treatment of samples with RNase A for 1 h at 37 degrees C and proteinase K overnight at 65 degrees C. DNA was purified using a PCR-purification kit (Qiagen). ChIP DNA was prepared for sequencing by blunting the DNA with a mixture of T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK (NEB) according to the manufacturer's instructions. dA overhangs were then added and Illumina adapters ligated. Adapter-ligated DNA was subject to 18 cycles of PCR before size selection by agarose gel electrophoresis. Amplified DNA was purified using the Qiaquick gel extraction kit (Qiagen). The purified DNA was quantified both with an Agilent Bioanalyzer and Invitrogen Qubit.
characteristics.characteristic: description
characteristics.text: MATa his3-delta200 leu2-delta0 lys2-delta0 trp1-delta63 ura3-delta0 met15-delta0 can1::MFA1pr-HIS3 hht1-hhf1::NatMX4 hht2-hhf2::[HHTS P16V-HHFS-KanMX6]-URA3
characteristics.characteristic: genotype
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_4932
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: scientific_name
characteristics.text: P16V H3K4me3 levels
characteristics.characteristic: title