title: KL007
organism: Mycosarcoma maydis
description: Protocols: Cells were harvested and quick frozen in liquid nitrogen. Briefly, strains (JB1, JB1 cib1-3xHA, UVO151 cib1-3xHA) were inoculated in CM supplemented with 1% glucose (CMG) overnight to an OD600 of 0.25. Cells were washed once with CM supplemented with 1% arabinose (CMA), resuspended in CMA for Clp1 expression (Pcrg1:clp1) and Tm was added to a final concentration of 5 µg/ml for UPR induction. ChIP analysis was done essentially as described before in Hampel et al. (2016). Briefly, strains (JB1, JB1 cib1-3xHA, UVO151 cib1-3xHA) were inoculated in CM supplemented with 1% glucose (CMG) overnight to an OD600 of 0.25. Cells were washed once with CM supplemented with 1% arabinose (CMA), resuspended in CMA for Clp1 expression (Pcrg1:clp1) and Tm was added to a final concentration of 5 µg/ml for UPR induction. After 4h of UPR induction, cells were fixed with formaldehyde (f.c. 1%) for 15 min at room temperature (RT). The reaction was quenched by addition of 2.5 M Glycin (f.c. 125 mM). Cross-linked cells were harvested by centrifugation and washed three times with TBS (50 mM Tris, 150 mM NaCl) and resuspended in 1,5 ml FA-Lysis Buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% (w/v) sodium deoxycholate, 0.1% SDS) supplemented with 1x cOmplete EDTA-free (Roche) protease inhibitor cocktail. Cells were shock-frozen in liquid nitrogen and disrupted in a cell mill (Retsch MM200, 25Hz, 5min). Chromatin was sheared in a Covaris S200 set to yield a DNA average size of approximately 100-300 bp. After centrifugation (17000g, 15 min, 4°C) 400 μl chromatin solution and 30 μl monoclonal Anti-HA-Agarose (clone HA-7, Sigma-Aldrich) were incubated overnight on a rotating wheel at 4°C. 50 μl of the chromatin was used as input control. The beads were washed three times with 500 μl FA lysis buffer, twice with 500 μl FA lysis high salt buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% (w/v) sodium deoxycholate, 0.10% SDS), twice with chromatin immunoprecipitation (ChIP) wash buffer (10 mM Tris-Cl pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet P40, 0.5% sodium deoxycholate), and once with 500μl TE (Tris-EDTA pH 7.5). The Protein-DNA complexes were eluted two times, once with 100μl ChIP elution buffer (50 mM Tris-Cl pH 7.5, 1 mM EDTA, 1% SDS) for 15 min at 65°C and once with 150 μl TE 0.67% SDS for 10 min at 65°C. The eluted samples were incubated over night at 65°C to reverse the crosslinks. After RNase A (0.8 mg/ml) incubation for 30 min at 37°C and Proteinase K (0.6 mg/ml) treatment for 2 h at 37°C DNA was recovered by column purification (PCR Purification Kit, Qiagen) and subjected to library preparation. For CHiP-Seq experiments the libraries were prepared using the NEBNext Ultra II DNA Library Prep with Beads as recommended by the manufacturer (New England BioLabs, Frankfurt am Main, Germany). To assess quality and size of the libraries samples were run on an Agilent Bioanalyzer 2100 using an Agilent High Sensitivity DNA Kit as recommended by the manufacturer (Agilent Technologies, Waldbronn, Germany). Concentration of the libraries were determined using the Qubit® dsDNA HS Assay Kit as recommended by the manufacturer (Life Technologies GmbH, Darmstadt, Germany).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-04-04T04:03:49Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-12-03T10:26:15Z
attributes.tag: External Id
attributes.value: SAMEA5145535
attributes.tag: INSDC center name
attributes.value: Philipps University Institute for Molecular Biology and Tumor Research
attributes.tag: INSDC first public
attributes.value: 2019-04-04T04:03:49Z
attributes.tag: INSDC last update
attributes.value: 2018-12-03T10:26:15Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7460:KL007
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: JB1 Pcrg:clp1; Cib1-3xHA
attributes.tag: sample name
attributes.value: E-MTAB-7460:KL007
attributes.tag: scientific_name
attributes.value: Ustilago maydis
attributes.tag: strain
attributes.value: JB1 (a1 b)