GSM6267786: sc_myc_pho4_nexus_2; Saccharomyces cerevisiae; OTHER
The yeast chromatin preparation was performed as previously described with minor modifications (Rossi, Lai, & Pugh, 2018). For each experiment, 50 ml of cells were cross-linked with 1% formaldehyde (Fisher Scientific) for 15 min at 25oC. The reaction was quenched by incubating with 125 mM glycine for 5 min. Cells were washed three times with cold wash buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl) at 4oC. The pellet was snap frozen in liquid nitrogen after the last wash. The pellet was then resuspended in 700µl of FA lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% triton-X-100, 0.1% Sodium deoxycholate, supplemented with protease inhibitor) and transferred to 1.5 ml of Sarstedt micro tube. 1 ml of 0.5 mm zirconia/silica beads was added to the tube and cells were lysed in a Mini-BeadBeater 24 (BioSpec products) for three cycles (3 min ON/5 min OFF).Samples were kept on ice during the OFF cycles. Cell lysates were separated from silica beads and collected in a new 1.5 ml tube using a 23G 1” needle (BD Microlance) and centrifuged at maximum speed for 4 min at 4oC to pellet the chromatin. The supernatant was discarded and the pellet was resuspended in 750 µl of FA lysis buffer supplemented with 0.1% SDS. Samples were sonicated with a Bioruptor Pico (Diagenode) for 18 cycles of 30 s ON and 30 s OFF intervals. Chromatin extracts were centrifuged at 16,000 g for 10 min at 4oC and the supernatant was used for the ChIP reaction. The ChIP-nexus was performed as previously described (He, Johnston, & Zeitlinger, 2015), with some modifications. Briefly, the chromatin was washed with Nexus wash buffers A to D (wash buffer A: 10 mM Tris-EDTA, 0.1% Triton X-100; wash buffer B: 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 5.2% sucrose, 1.0% Triton X-100, 0.2% SDS; wash buffer C: 250 mM NaCl, 5 mM Tris-HCl, pH 8.0, 25 mM HEPES, 0.5% Triton X-100, 0.05% sodium deoxycholate, 0.5 mM EDTA; wash buffer D: 250 mM LiCl, 0.5% IGEPAL CA-630, 10 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 10 mM EDTA) and 10 mM Tris, pH 7.5. End-repair and dA-tailing enzymatic reactions were performed using the NEBNext End Repair and NEBNext dA-tailing module. ChIP-nexus adapter mix containing four fixed barcodes (ACTG, CTGA, GACT and TGAC) were ligated with quick T4 DNA ligase. Following barcode extension, samples were treated with lambda exonuclease to generate high-resolution transcription factor binding footprints. Reverse crosslinking was performed at 65C for overnight, followed by ethanol precipitation of DNA. Purified DNA fragments were circularized, and directly subjected to PCR amplification. The libraries were gel purified before sequencing. For each factor, at least two biological replicates were prepared side-by-side from independent colonies.