GSM1848231: Hst1_ChIPSeq; Clavispora lusitaniae; ChIP-Seq
Crosslinked cell pellets were stored at -80°C. For lysis, cells were suspended in 100 µl ice cold lysis buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 mM TPCK, 1mM PMSF, 5 µg/mL chymostatin, 2 µg/mL pepstatin A, 1 mM benzamidine, and 1× complete protease inhibitors; Boehringer Mannheim) and vortexed with glass beads for 30 minutes at 4°C. The volume was brought to 800 µl and transferred away from the beads to fresh tubes. The lysate was sonicated four times for 10 seconds at setting 10 on a Sanyo Soniprep 150, and spun for 10 minutes in a microfuge at 4°C. The supernatant was collected and 160 µl was used in a final volume of 400 µl lysis buffer for each immunoprecipitation reaction. Samples were pre-cleared for 30 min with 30 µL of a 50% slurry of Protein A agarose beads and then incubated overnight with 5 µL of antibody (α-myc rabbit (Millipore-06-549).  Samples were then incubated with 10 μL Protein A agarose beads for 1 h. The beads were washed for five minutes in the following buffers:  wash 1 (150 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), wash 2 (500 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), wash 3 (0.25 M LiCl, 10 mM Tris pH 8.0,1 mM EDTA, 1% sodium deoxycholate, 1% NP40), wash 4 (10mM Tris pH 8.0, 1mM EDTA) and wash 5  (10mM Tris pH 8.0, 1mM EDTA, 20µg of RNaseA). Material was eluted from beads in 500 μL 0.1 M NaHCO3, 1% SDS. Cross-links were reversed by overnight incubation at 65°C.  DNA was then extracted with phenol:chloroform and precipitated with ethanol. Illumina standard sequencing protocol
GEO
GSM1848231