title: GSM1144378: Total 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 total nucleosomes, one 150 μL from each reaction was treated with 38 μL of 5xTE/SDS (50mM Tris pH8, 5mM EDTA, 5% SDS) overnight at 65˚C to reverse the crosslink. The remaining of the extract was kept as a backup at -20˚C. The next day, 210 μL of RNase mix (205 μL TE, 3 μL RNaseA, 2 μL glycogen) was added, followed by an incubation of two hours at 37˚C. 8 μL of proteinase K (from 20mg/mL stock) was added and incubation was pursued for an additional two hours at 37˚C. DNA was purified by phenol:chloroform extraction, EtOH precipitated and resuspended in 50 μL of 10mM Tris pH 8.0. Inspection on agarose gel revealed some residual RNA so an additional RNaseA treatment, followed by phenol:chloroform extraction and EtOH precipitation was performed. Aliquots were inspected on 2% agarose gel and the sample contains mainly monononucleosomes. 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: GSM1144378