characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-21T16:00:06Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-21T16:00:06Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112156533
characteristics.characteristic: External Id
characteristics.text: Masaryk University, NCBR
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-21T16:00:06Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-21T16:00:06Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14266430
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12401:IN_NKG1 no tag
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: fission yeast
characteristics.characteristic: common name
characteristics.text: Protocols: All strains were cultivated into the mid-log phase (OD = 0.4 - 0.6) and incubated with 1 % formaldehyde for 15 min at room temperature to cross-link DNA-protein complexes. Glycine was added to a final concentration of 125 mM, and the incubation continued for 5 min. 5x10^8 cells were harvested and washed with 10 ml of ice-cold PBS. The yeast cell wall breakage was performed in 400 ul CHIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, Complete EDTA-free protease inhibitor cocktail tablets, Roche) with half the volume of glass beads in 2 ml low binding tubes using FASTprep-24. The suspension was washed two times with CHIP lysis buffer (15 min, 4˚C, 15000 rpm), and 300 ul of the extract was sonicated with Bioruptor (Diagenode, 30 sec ON/30 sec OFF, High Power, 25 times) and clarified by centrifugation (15 min, 4˚C, 15000 rpm), resulting in an average DNA fragment size of 300-500 bp. 5 ul of the sonicated precleared extract was taken as an input control sample. Monoclonal anti-FLAG M2 antibody (F1804, Sigma) was diluted 1:150, incubated with precleared cell extract in 1.5 ml low binding tube for 2 h on ice, and precipitated over-night with Dynabeads protein G (Invitrogen). Precipitates were washed with 1 ml of CHIP lysis buffer, 1 ml of High Salt buffer (CHIP lysis buffer with 500 mM NaCl), 1 ml of Wash buffer (10 mM Tris-HCl at pH 8.0, 0.25 M LiCl, 0.5 % NP-40, 1 mM EDTA), and 1 ml of TE buffer (20 mM Tris-HCl at pH 8.0, 1 mM EDTA). Elution (50 mM Tris at pH 8, 0.1 % SDS, 10 mM EDTA) and de-crosslinking of samples were done overnight at 65˚C. DNA was purified using QIAquick PCR Purification Kit (Qiagen). The input DNA samples were tested for DNA fragmentation and determination of DNA concentration by the Fragment analyzer (Agilent). Input and immunoprecipitation (IP) samples with the best fragmentation and high concentration were used for the creation of NGS libraries (NEBNEXT ULTRA II DNA Library Prep kit, NEB).
characteristics.characteristic: description
characteristics.text: ura4-D18, ade6-704, leu1-32
characteristics.characteristic: genotype
characteristics.text: asynchronous culture
characteristics.characteristic: growth condition
characteristics.text: Schizosaccharomyces pombe
characteristics.characteristic: organism
characteristics.text: Schizosaccharomyces pombe
characteristics.characteristic: organism
characteristics.text: whole organism
characteristics.characteristic: organism part
characteristics.text: G1
characteristics.characteristic: replicate
characteristics.text: Schizosaccharomyces pombe
characteristics.characteristic: scientific_name
characteristics.text: 501
characteristics.characteristic: strain
characteristics.text: IN_NKG1 no tag
characteristics.characteristic: title