GSM3105111
GSM3105111: 20170921_run55_universal_R64-1-1_13; Saccharomyces cerevisiae; MNase-Seq
Recombinant remodeler were purified via affinity tag purification and ion exchange chromatography. The yeast genome plasmid library used was derived from Sa3A digested yeast genome fragments and ligated via BamHI into the YCp50 plasmid. Chromatin was reconstituted via salt gradient dialysis. Remodeling reactions were started with adding 10 nM of protein to 90 nM chromatin and stopped via apyrase treatment. After MNase treatment, mononucleosomes were isolated and sequencing libraries prepared according to NEBNext Multiplex Oligos for Illumina protocols. The NEBNext Multiplex Oligos for Illumina were used to generate sequencing libraries. Approximately 50 ng of purified mononucleosomal DNA after MNase digest 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 min at 12°C, 15 min at 37°C and 20 min at 72°C. To ligate NEBNext Adaptors (0.75 µM final concentration, NEB) to the DNA, samples were incubated with T4 DNA ligase (NEB) at 25°C for 10 min, followed by incubation with 2 U USER enzyme (NEB) for 10 min at 37°C. Fragments were purified using AMPure XP beads (Beckman Coulter) and amplified for 10-12 cycles using NEBNext Universal Primer and NEBnext Index Primer, 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 10 s, 65°C for 30 s, 72°C for 30 s (repeated for 12 cycles) with a final amplification step at 72°C for 5 min. The Qubit dsDNA HS Assay Kit (Invitrogen) was used to assess the DNA content of 1 µL PCR reaction. Adaptor-ligated mono-nucleosomal DNA was purified from agarose gels via Freeze N Squeeze DNA Gel Extraction Spin Columns (Biorad), ethanol precipitated and resuspended in 15 µL 0.1x TE buffer. DNA was measured 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). 10 nM samples were pooled according to sequencing lane requirements. The final pool was quantified by BioAnalyzer (Agilent) and analyzed on an Illumina HiSeq 1500 in the 50 bp single-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).
GEO
GSM3105111
GEO Accession
GSM3105111