Schizosaccharomyces pombe
Protocols: For Cbf11-related analyses, 400 mL cultures of Cbf11-TAP (MP707), Cbf11DBM-TAP (MP712), Cbf11-TAP_mga2KO (MP921), TAP-Mga2 (MP872), TAP-Mga2_cbf11KO (MP915), untagged WT (JB32, a negative control) and Fkh2-TAP (MP931, forkhead transcription factor unrelated to CSL; a negative control) cells were grown in YES to exponential phase. For Cbf12-related analyses, 10-15 mL of Cbf12-TAP (MP540), Cbf12DBM-TAP (MP518), untagged WT (JB32) and Fkh2-TAP (MP931) cells were grown in YES to stationary phase and were brought up to 400 mL with YES before fixation. Three independent biological experiments were performed for the CSL proteins; the negative controls were grown once for each cultivation condition. Saccharomyces cerevisiae cells expressing the histone H2A.2-TAP (Euroscarf yeast strain collection, original ID SC0421, lab ID MP792) were used as a potential spike-in control for normalization of ChIP samples of TAP-tagged S. pombe proteins. S. cerevisiae cells were cultivated to exponential phase (OD600 1.8) in the complex YPAD medium at 30°C, and chromatin extract preparation was performed as described below for S. pombe with these exceptions: fixation for 30 min, 15 cycles of chromatin shearing. However, after assessing the performance of the spike-in control, we decided not to take it into account during subsequent data analyses. Cells were fixed by adding formaldehyde to the final concentration of 1%. After 10 min incubation, the remaining formaldehyde was quenched by 125 mM glycine for 10 min. Cells were washed with PBS and broken with glass beads in the Lysis Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, FY protease inhibitors [Serva], 1 mM PMSF) using the FastPrep24 machine (MP Biomedicals). Extracted chromatin was sheared with the Bioruptor sonicator (Diagenode) using 20 cycles of 30 s on, 30 s off at high power settings. An aliquot of each chromatin extract was kept as the input control sample (used for a qPCR validation step), and 1.5-2 mg of chromatin extract with added S. cerevisiae spike-in chromatin (40,000-fold dilution) were used for immunoprecipitation with 100 μl of BSA-blocked magnetic IgG-coated beads (Invitrogen, 110.41). Chromatin immunoprecipitation was performed on a rotating wheel overnight at 4°C. The precipitated material was ChIP-exo treated according to the updated, simplified version of the ChIP-nexus approach (ChIP-nexus [version 2019]; https://research.stowers.org/zeitlingerlab/protocols.html) introduced by (He et al. 2015), except for wash buffers A-D that were optimized for yeasts as previously described (Doris et al. 2018). ChIP-exo treated material was eluted in Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). Cross-links were reversed at 65°C for 6 h, samples were treated with DNase-free RNase A followed by proteinase K digestion, and purified using standard phenol-chloroform extraction and sodium acetate/ethanol precipitation. qPCR was performed to check the efficiency of ChIP. ChIP-nexus library construction for Illumina sequencing was performed as described in the updated version of the ChIP-nexus approach (ChIP-nexus [version 2019]; https://research.stowers.org/zeitlingerlab/protocols.html). Concentration of ChIP-nexus libraries was measured using the Quantus fluorometer (Promega) and fragment size distribution was checked on Agilent Bioanalyzer using the High Sensitivity DNA Assay.
ENA first public
2024-04-05
External Id
SAMEA114263916
INSDC center name
Charles University, Faculty of Science Department of Cell Biology
INSDC last update
2023-08-14T13:30:06Z
INSDC status
public
Submitter Id
E-MTAB-13258:B28
broker name
ArrayExpress
collection date
not collected
common name
fission yeast
developmental stage
exponential phase
genotype
Cbf11-TAP_mga2KO
geographic location (country and/or sea)
not collected
growth condition
YES medium
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-13258:B28
scientific_name
Schizosaccharomyces pombe
spike in
added
strain
MP921