title: Input_Kin28WT
organism: Saccharomyces cerevisiae S288C
description: Protocols: 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 µm, Sigma) are then added to each tube, and the yeast cells were lysed at 4°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°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 incubate on a rotating wheal at 4°C for 1 to 2 hours.Spin down 20 min / 12000 rpm / 4°C. Vacuum the supernatant and resuspend in 1.6 ml FA/SDS/PMSF buffer with a burned Pasteur pipet. Sonication step was performed on a S220 focused-ultrasonicator (Covaris) in 1mL milliTube (Covaris), for two cycles of 3 minutes spaced by a 30s rest time. Each cycle consisted of 150W pulses for 10% of the time (duty factor 10). Sonicated lysates were then transferred to a new 2mL safe-lock tube and centrifugation was performed at 15000g for 20 minutes. 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°C. Spin down 30 min / 10000 rpm / 4°C on a JA20 rotor (Beckmann).Transfer the supernatant in a new tube. Aliquot the supernatant (the chromatin is now soluble). Aliquots of 650 µl and freeze in liquid nitrogen.Chromatin fragment control: Take an aliquot of 100 µl of chromatin and add 25 µl Pronase buffer 5X and 6.25 µl of Pronase (20 mg/ml in H20). Incubate 1 hour at 37 °C and O/N at 65 °C (digestproteins and reverse crosslink). Add 3.5 µl of RNaseA 1mg/ml and incubate 1 hour at 37 °C. Purify on a Quiagen PCR purification column (Elute wih 50 µl Tris 10 mM pH8.5). Load on gel (1.5% in TAE -100V).Immunoprecipitation:Wash 50 µl of Dynabeads Ig 4 times with 500 µl PBS BSA 0.1% buffer. Resuspend in 100 µl PBS BSA 0.1 % buffer. Add antibody. Incubate 30 min at 1300 rpm in an Eppendorf shaker at 30 °C. Wash 2X 500 µl PBS BSA 0.1%. Wash 1X 500 µl PBS BSA 0.1% with shaking 10 min at 1300 rpm / 30 °C. Wash 1X 500 µl PBS BSA 0.1%.Spin down the chromatin from the -80°C for 15 min / 12000 rpm / 4 °C. Add 50 µl of PBS BSA 10mg/ml to the beads and then 500 µl of chromatin.Incubate 2h at 21 °C in an Eppendorf shaker (1300 rpm). Resuspend with 500 µl 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 °C. Wash 1X with 500 µl IP buffer (Tris pH7.5 125 mM, EDTA 25 mM, SDS 2.5%). Wash 1X with 500 µl TE. Elute with 125 µl Pronase Buffer 1X for 20 min at 65 °C in an Eppendorf shaker at 600 rpm. Cool down at RT. Add 6.25 µl Pronase to the supernatant. Incubate 1h at 37 °C and O/N at 65 °C. Add 25 µl of Pronase buffer 5X and 6.25 µl of Pronase to 100 µl of chromatin remaining from the initial tube (total DNA).Incubate 1h at 37 °C and O/N at 65 °C. Add 3.5 µl RNAseA (1mg/ml) and incubate 1h at 37 °C. Following a 75 minutes shift at 37°C, after reaching exponential phase at 25°C in YPD, cells were cross-linked with 1% formaldehyde during 10 minutes. DNA was purified on a Qiagen PCR purification column Libraries were constructed with TruSeq ChIP library preparation following manufacturer recommandations.