title: GSM1144379: H2A.Z mononucleosomes; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: One cell pellet was kept as a backup and the other one was thawed and resuspended in 20 mL of buffer Z (1M Sorbitol, 50mM Tris pH 7.4) before adding 14 μL of β-mercaptoEtOH and 0.5 mL of zymolase solution (1mg/mL, freshly prepared in buffer Z). Cells were shaken for 40 minutes at 30˚C and centrifuged 10 minutes at 1,500 rpm. The supernatant was aspired and discarded while the pellet was resuspended in 1.25 mL of NP buffer (50mM NaCl, 10mM Tris pH7.4, 5mM MgCl2, 1mM CaCl2, 0.075% NP40, 0.5mM spermidine, 1mM β-mercaptoEtOH). After this one, one of two different protocols was applied. For H2A.Z nucleosomes, 40 μL of 500mM HEPES pH7.5, 22,4 μL of NaCl 5M, 16 μL of 5% Na-deoxycholate, 80 μL of 10% TritonX-100 and a protein inhibitor cocktail were added to the extracts. H2A.Z was immunoprecipitated overnight at 4˚C with agitation using 50 uL magnetic beads (Dynal) coupled to 2 μg of anti-Myc antibody 3E10 (prepared in-house) with rotation (note that the yeast strain used has a 3xMyc-tagged HTZ1 gene). The beads were then processed as described before (PMID 19378172), inspected on agarose gel as described above and the sample contains mainly H2A.Z mononucleosomes. ChIP samples were prepared from yeast cells. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature, the reaction was quenched by 0.125 M glycine, and the cells were washed in PBS and harvested in the presence of protease inhibitors. ChIP was performed as described in Robertson et al. 2008. Briefly, chromatin DNA was fragmented and chromatin was pre-cleared with 40µl of blocked Protein A/G sepharose beads (Amersham, USA) at 4oC for 2 hrs, supernatants were removed from the beads following centrifugation, and transferred to fresh tubes. Sepharose beads were used for ChIPs and each immunoprecipitation was carried out with pre-cleared chromatin and 5 μg of antibody (anti-Myc 9E10) and incubated at 4oC for 1 hr. To each immunoprecipitation reaction, 20 µl of Protein A/G beads were added and incubated by rotating at 4oC overnight. Beads were recovered by centrifugation and washed twice with ChIP wash buffer (20 mM Tris-HCl [pH 8.0], 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl) and once with ChIP final wash buffer (20 mM Tris-HCl [pH 8.0], 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl). DNA-antibody complexes were eluted using 100uL Elution Buffer (100 mM NaHCO3, 1% SDS), and 5 µg of DNase-free RNase (Roche, Canada) was added and incubated at 68°C for 2 hours with shaking on a thermomixer to reverse the crosslink. The beads were pelleted by centrifugation and the supernatant was collected. Elution was repeated with the addition of 100 µl of Elution Buffer and incubation at 68oC for 5 min with shaking on a thermomixer. After pooling the two elutions, DNA was recovered from the eluate using the QIAquick PCR Purification kit (Qiagen, Germany). ChIP DNA was run and size separated by PAGE (8%). The 100-300bp DNA fraction was excised and eluted from the gel slice overnight at 4°C in 300 μl of elution buffer (5:1, LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA)-7.5 M ammonium acetate), and was purified using a Spin-X Filter Tube (Fisher Scientific), and by ethanol precipitation. The library was prepared by following the paired-end library protocol (Illumina Inc., USA). Briefly, the DNA was subject to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, and then 3' A overhangs generation by Klenow fragment (3' to 5' exo minus), and ligated to Illumina PE adapters, which contain 5' T overhangs. The adapter-ligated products were purified on QIAquick spin columns (Qiagen), then PCR-amplified with Phusion DNA Polymerase in 10-15 cycles using Illumina's PE primer set (Illumina). PCR product of desired size range was purified using a 8% PAGE, and the DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay and Nanodrop 7500 spectrophotometer (Nanodrop, USA) and diluted to 10nM. The final concentration was double checked and determined by Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen). Clusters were generated on the Illumina cluster station and sequence was run on an Illumina Genome Analyzer IIx following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM1144379