GSM4144980: bre1Δ V5 ChIP repeat 2; Saccharomyces cerevisiae; ChIP-Seq
S. cerevisiae strain YZS375 transformed with pMC6-based constructs to express either Rad6-3V5 or the mutants (rad6-P43L-3V5, rad6-E49K-3V5 or rad6-P47T-3V5) or strain YZS413 (bre1Δ) transformed with vector pRS314 were grown in –TRP medium to mid-log phase, and cross-linked with 1% formaldehyde (Sigma) for 20 min at room temperature. Crosslinking of cells was stopped by adding glycine (0.125M) and washed once with PBS. Multiple aliquots of 100 x 107 cross-linked cells were harvested. For the reference or spike-in genome, C. glabrata transformed with pMC8 was grown in YPAD medium supplemented with cloNAT (100 µg/ml), subjected to formaldehyde crosslinking as described above and aliquots of 20 x 107 cells were harvested. Cross-linked S. cerevisiae (100 x 107) and C. glabrata (20 x 107) cells were combined prior to spheroplasting, followed by nuclei isolation and chromatin preparation as previously described. Briefly, nuclei were resuspended in FA140 buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl) and sonicated in Diagenode Bioruptor® Pico for 30 min (60 cycles of 30 sec ON and 30 sec OFF). After centrifugation (13,200 rpm, 15 min at 4°C), an aliquot of the dialysate (25 μl) was set aside for isolating input DNA. Prior to immunoprecipitation, chromatin was precleared with 50μl Protein A or Protein G Dynabeads (Invitrogen) blocked with bovine serum albumin and fish skin gelatin. Pre-cleared chromatin was then combined with anti-V5 antibody (15 μl; Bio-Rad, Catalog No. MCA1360) or custom anti-Bre1 antibody (15 μl), incubated overnight at 4°C with end-over-end rotation. The immune-complex was captured by adding 50 μl Protein A or Protein G Dynabeads and incubating for an additional 2.5 h. Bead-bound complexes were then washed sequentially for 5 min each with end-over-end rotation at 4°C with FA buffer+140 mM NaCl (3 washes), FA buffer+1000 mM NaCl (2 washes), FA buffer+500 mM NaCl (2 washes), and one wash each with LiCl/NP40 buffer (10 mM Tris.Cl, pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate) and TE (10 mM Tris.Cl, pH 8.0, 1 mM EDTA). Elution, reverse crosslinking of ChIP and input DNA, and enzymatic digestion to remove RNA or protein were performed as described. Library construction was performed using NEBNext Multiplex Oligos (Index Primers Set 1 or Set2) and NEBNext ChIP-seq Library Prep Reagent Set for Illumina prior to 50 bp single-end sequencing using Illumina Hiseq2500 NEBNext ChIP-Seq Library Prep Reagent Set for Illumina NEB's Index Primer Set1 and Primer Set 2
GEO Accession
GSM4144980