GSM4294700: MPS1_32°C_InPut; Saccharomyces cerevisiae; ChIP-Seq
100 mL of cells (concentration 107cells/mL) were fixed in 1% formaldehyde for 30' at RT with shaking and then transferred ON at 4°C. Cells were then spun down 3min at 3000 rpm at 4°C and washed three times with TBS. Cells were resuspended in 0.4 mL of 50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate containing a cocktail of protease inhibitors (Complete EDTA-free Roche) and 1mM PMSF, followed by cell breakage through a Multi-beads shaker (Yasui-Kikai) at 0°C. Lysates were eluted in 15mL Falcon tubes (centrifuged twice at 3000 rpm for 1 min at 4°C), transferred in 1.5 Eppendorf tube, centrifuged at 5000 rpm 1 min at 4°C and sonicated through a Sonifyer Branson 2508 (5-7 cycles of 15sec, tune 1.5 with centrifugation of 5000 rpm for 1 min between each cycle). Sonicated lysates were then centrifuged for 5 min at 15000 rpm at 4°C. Supernatants were recovered. 10 µL of lysates were withdrawn at this stage (=Inputs). For immunoprecipitations (IPs), 60 µL of Protein G-dynabeads pre-washed twice with PBS containing 5 mg/mL BSA and pre-adsorbed with 3 µg Babco 16B12 anti-HA antibody were added to each lysate and incubated ON at 4°C on a nutator. IPs were then washed twice with ice-cold 50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate, twice with ice-cold 50mM Hepes-KOH pH7.5, 500mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate, twice with ice-cold 10mM Tris-Cl pH8, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA and once with ice-cold TE (50mM Tris-Cl pH8, 1mM EDTA pH8). IPs were eluted at 65°C for 15min with 50µL of 50mM Tris-Cl pH8, 10mM EDTA, 1% SDS, resuspended in 50 µL of TE/1% SDS and incubated overnight at 65°C to reverse crosslinking. IPs were then incubated at 37°C for 1 hour with 100 µL of TE containing 10 µg of RNAseA and 2 hours at 37°C with 2 µL of ProteinaseK 50 mg/mL. DNA was then purified with the Qiagen PCR purification kit Libraries for Illumina sequencing have been prepared following the NEBNext Ultra II DNA Library Prep Kit for Illumina.
GEO Accession
GSM4294700