title: IP_G5delF3
organism: Schizosaccharomyces pombe
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-02-21
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-02-21
attributes.tag: External Id
attributes.value: SAMEA112156538
attributes.tag: INSDC center alias
attributes.value: Masaryk University, NCBR
attributes.tag: INSDC center name
attributes.value: Masaryk University, NCBR
attributes.tag: INSDC first public
attributes.value: 2023-02-21T16:00:06Z
attributes.tag: INSDC last update
attributes.value: 2023-02-21T16:00:06Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12401:IP_G5delF3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fission yeast
attributes.tag: genotype
attributes.value: Δgcn5::G418, Nse4-FLAG-His::KanR, ura4-D18, ade6-M216?, leu1-32
attributes.tag: growth condition
attributes.value: asynchronous culture
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: replicate
attributes.value: F3, biological replica
attributes.tag: sample name
attributes.value: E-MTAB-12401:IP_G5delF3
attributes.tag: scientific_name
attributes.value: Schizosaccharomyces pombe
attributes.tag: strain
attributes.value: YLJ375