title: GSM4306348: Replicate1_hs_Reb1-1_3_INO80; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: MNase-seq  After apyrase addition, remodeling reactions were supplemented with CaCl2 to a final concentration of 1.5 mM and digested with 100 U MNase to generate mostly monoucleosomal DNA. Chd1-reaction were incubated with 20 U MNase to get the same extent of mononucleosomal DNA. 10 mM EDTA and 0.5% SDS (final concentrations) were added to stop the MNase digest. After proteinase K treatment for 30 min at 37°C, samples were ethanol precipitated and electrophoresed for 1.5 - 2 h at 100 V using a 1.5% agarose gel in 1x Tris-acetate-EDTA (TAE) buffer. Mononucleosome bands were excised and purified with PureLink Quick Gel Extraction Kit. For library preparation, approximately 10 - 50 ng of mononucleosomal DNA was incubated with 1.25 U Taq polymerase (NEB), 3.75 U T4 DNA polymerase (NEB) and 12.5 U T4-PNK (NEB) in 1x ligation buffer (B0202S, NEB) for 15 minutes at 12 °C, 15 minutes at 37 °C and 20 minutes at 72 °C. To ligate NEBNext Adaptors (0.75 µM final concentration, from NEBNext Multiplex Oligos Kit) to the DNA, samples were incubated with T4 DNA ligase (NEB) at 25 °C  for 15 minutes, followed by incubation with 2 U USER enzyme (NEB) for 10 minutes at 37°C. Fragments were purified using 2 volumes AMPure XP beads (Beckman Coulter) and amplified for 8-10 cycles using NEBNext Multiplex Oligos, Phusion High-Fidelity DNA Polymerase (1 U, NEB), deoxynucleotide solution mix (dNTP, 2.5 mM, NEB) and Phusion HF Buffer (1x, NEB). The following protocol was applied for amplification: 98 °C for 30 s, 98°C for 10s, 65 °C for 30s, 72 °C for 30s with a final amplification step at 72 °C for 5 minutes. The Qubit dsDNA HS Assay Kit (Invitrogen) was used to assess the DNA content of 1 µL PCR reaction. DNA was purified from agarose gels via PureLink Quick Gel Extraction Kit, measured again with Qubit dsDNA HS Assay Kit and diluted to final concentration of 10 nM (calculation based on the assumption that the DNA fragment length is 272 bp, i. e., 147 bp nucleosomal DNA and 122 bp sequencing adaptor). Diluted samples were pooled according to sequencing reads (~6 Mio reads/ sample). The final pool was quantified by BioAnalyzer (Agilent) and analyzed on an Illumina HiSeq 1500 in the 50 bp single-end or paired-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).
attributes.tag: GEO Accession
attributes.value: GSM4306348