characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-01-15T17:01:43Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:09:36Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2779897
characteristics.characteristic: External Id
characteristics.text: European Molecular Biology Laboratory
characteristics.characteristic: INSDC center name
characteristics.text: 2015-01-15T17:01:43Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:09:36Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS549647
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2945:sc_htchip_set2rtt109_WCE_3
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: Cells were grown in 50 mL YPD (1% yeast extract, 2% peptone, 2% glucose and 40 mg adenine) and harvested at an OD600 of ~0.5. alpha-factor was added to reach a final concentration of 50ng/mL. Cells were then grown for two generations before harvest. Cross-linking was performed with 1% formaldehyde (final concentration) for 15 min at room temperature on a shaker plate. Fixation was quenched with the addition of Glycine to a final concentration of 0.135M. Cross-linked cells were washed three times in ice-cold TBS buffer (20 mM Tris pH 7.5, 0.146 M NaCl). Yeast pellets were resuspended in NP-S buffer (0.5 mM spermidine, 0.075% NP40, 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 1mM ??-mercaptoethanol) with Protease Inhibitor Cocktail (Roche) and lysed at 4??C with 2 volumes of glass beads in a FastPrep instrument (MP Biomedicals) using the following settings: 4 x 20 sec at 6.5 m/sec with 1 min pause between each pulse. Nucleosome fractions were isolated by incubating lysates with 80U of Micrococcal Nuclease (Worthington Biochemicals) for 45 min at 37??C. EGTA was added to samples at a final concentration of 0.01M for enzyme inactivation. Antibodies were diluted in PBS containing 5 mg/mL BSA (Sigma) and 10-30 microg of yeast tRNA (Ambion) and conjugated to 1.5 mg and 4.5 mg of protein A Dynabeads (Invitrogen) for classical ChIP and HT-ChIP respectively, for 4 hrs at 4C on a wheel. 50 microg of non-barcoded nucleosomes (classical ChIP) were diluted in lysis buffer (50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1 % Sodium Deoxycholate, 1 mM Benzamidin and Protease Inhibitor Cocktail) and incubated with antibody-coated magnetic beads overnight at 4C on a wheel. Chromatin-antibody complexes were washed 8 times in RIPA buffer (0.05 M Hepes, 0.5 M LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Sodium Deoxycholate) and chromatin was eluted from the antibody-coated beads by two 10 min-incubations in ChIP elution buffer (50 mM Tris pH 8.0, 10 mM EDTA and 1% SDS) at 68C with mixing. Formaldehyde crosslinking, Cell lysis using Fast-prep, Micrococcal nuclease digestion, Chromatin IP, Reverse crosslinking and library preparation 20 to 100 ng of IP- and input DNA were end-repaired, dA-tailed and ligated to paired-end adapters as described above. Purification of the newly barcoded DNA samples was performed using Ampure XP beads. 1 ng of barcoded IP- and input DNA from BAR- and classical ChIP experiments was amplified by PCR using 0.4 ??L of each Illumina Paired-End primers (at 10 ??M) and 20 ??L of 2X Phusion HF Master Mix (New England Biolabs) with the following program: 30 sec at 98 ??C for initial denaturation, 30 sec at 98??C, 30 sec at 65??C, 30 sec at 72??C for 18 cycles, followed by 5 min at 72??C for final extension
characteristics.characteristic: description
characteristics.text: gene knock out
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000506
characteristics.characteristic: genetic modification
characteristics.text: BY4741_delta_set2_rtt109
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: mating type a
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001275
characteristics.characteristic: sex
characteristics.text: BY4741
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000098
characteristics.characteristic: strain
characteristics.text: sc_htchip_set2rtt109_WCE_3
characteristics.characteristic: title