characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-09-04T17:01:29Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:02:03Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2785610
characteristics.characteristic: External Id
characteristics.text: CEA, Evry
characteristics.characteristic: INSDC center name
characteristics.text: 2015-09-04T17:01:29Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:02:03Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS555300
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2961:Rad3-WT-2-IP_2
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: All strains were grown in YPD rich medium at 30 degrees C to 0.6 OD600, then shifted at 37 degrees C for 45min. The cultures were cross-linked with 1% formaldehyde for 10 min The cell pellets are resuspended in 1 ml of FA/SDS/PMSF buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) and transferred to 1.5 mL Eppendorf tubes. 0.75 mL glass beads (425-600 um, Sigma) are then added to each tube, and the yeast cells were lysed at 4 degrees C for 15 min by vortexing. Pierce the bottom of a 12 ml Greiner tube with a hot needle (0.7x30 mm or 0.5x15 mm) and place in a Falcon50 with a pierced cap. The lysates are transfered in the pierced tubes. Wash each Epp with twice 1 ml of FA/SDS/PMSF buffer (4 ml per extract). Spin down 1 min/2000 rpm. Gently resuspend the pellet with a 5 ml pipet and transfer in a sterile 15 ml Corex tube. Spin down 20 min / 12000 rpm / 4 degrees C in a JA17 rotor (Beckmann). Vacuum the supernatant. The cross-linked chromatin appears as a transparent layer around the pellet. Transfer the pellet in a 2 ml Epp with a burned Pasteur pipet. Add 0.8 ml of FA/SDS/PMSF buffer in the Corex and resuspend with the Pasteur pipet. Transfer in the Epp. Wash the Corex with 0.8 ml FA/SDS/PMSF buffer. Homogenize the Epp with the Pasteur pipet and ihncubate on a rotating wheal at 4 degrees C for 1 to 2 hours.Spin down 20 min / 12000 rpm / 4 degrees C. Vacuum the supernatant and resuspend in 1.6 ml FA/SDS/PMSF buffer with a burned Pasteur pipet.Place the tubes in a mix water / ice / NaCl. Sonicator settings: Pulse-60%-Power 4. Sonicate 3 times 40 sec with 20 sec intervals. Split in 200ul aliquots and performe an additional sonication step with Bioruptor (Diagenode), 6 cycles of 30s with medium intensity setting. Average size of fragments obtained is 200bp.Transfer in a 12 ml Greiner tube and add 0.5 ml FA/SDS/PMSF buffer. Incubate on a rotating wheal for 30 min to 1 hour at 4 degrees C. Spin down 30 min / 10000 rpm / 4 degrees C on a JA20 rotor (Beckmann).Transfer the supernatant in a new tube. Aliquot the supernatant (the chromatin is now soluble). Aliquots of 650 ul and freeze in liquid nitrogen.Chromatin fragment control: Take an aliquot of 100 ul of chromatin and add 25 ul Pronase buffer 5X and 6.25 ul of Pronase (20 mg/ml in H20). Incubate 1 hour at 37 degrees C and O/N at 65 degrees C (digestproteins and reverse crosslink). Add 3.5 ul of RNaseA 1mg/ml and incubate 1 hour at 37 degrees C. Purify on a Quiagen PCR purification column (Elute wih 50 ul Tris 10 mM pH8.5). Load on gel (1.5% in TAE-100V).Immunoprecipitation:Wash 50 ul of Dynabeads Ig 4 times with 500 ul PBS BSA 0.1% buffer. Resuspend in 100 ul PBS BSA 0.1 % buffer. Add antibody (5ul 8WG16 or 1ul 12CA5). Incubate 30 min at 1300 rpm in an Eppendorf shaker at 30 degrees C. Wash 2X 500 ul PBS BSA 0.1%. Wash 1X 500 ul PBS BSA 0.1% with shaking 10 min at 1300 rpm / 30 degrees C. Wash 1X 500 ul PBS BSA 0.1%.Spin down the chromatin from the -80 degrees C for 15 min / 12000 rpm / 4 degrees C. Add 50 ul of PBS BSA 10mg/ml to the beads and then 500 ul of chromatin.Incubate 2h at 21 degrees C in an Eppendorf shaker (1300 rpm). Resuspend with 500 ul FA/SDS and change tube. Wash 2X 1 ml FA/SDS + NaCl (final concentration 500 mM). Wash 1X 1 ml FA/SDS + NaCl with shaking 10 min at 1300 rpm / 21 degrees C. Wash 1X with 500 ul IP buffer (Tris pH7.5 125 mM, EDTA 25 mM, SDS 2.5%). Wash 1X with 500 ul TE. Elute with 125 ul Pronase Buffer 1X for 20 min at 65 degrees C in an Eppendorf shaker at 600 rpm. Cool down at RT. Add 6.25 ul Pronase to the supernatant. Incubate 1h at 37 degrees C and O/N at 65 degrees C. Add 25 ul of Pronase buffer 5X and 6.25 ul of Pronase to 100 ul of chromatin remaining from the initial tube (total DNA).Incubate 1h at 37 degrees C and O/N at 65 degrees C. Add 3.5 ul RNAseA (1mg/ml) and incubate 1h at 37 degrees C. Purify DNA on a Qiagen PCR purification column.
characteristics.characteristic: description
characteristics.text: tagging
characteristics.characteristic: genetic modification
characteristics.text: MATa ura3-52 his3-delta200 ade2-101uaa trp1-delta63 lys2-801uag leu2-delta1 RAD3::3HA::HIS3 med17::KanMX6 // MED17 CEN TRP1
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: Rad3-WT-2-IP_2
characteristics.characteristic: title