GSM1646416: abo1_mono_nucleosomes; Schizosaccharomyces pombe; MNase-Seq
Reactions were incubated at 65°C overnight with 0.2 mg/ml proteinase K and 10 μg RNAse. DNA was purified by extracting twice with phenol:chloroform followed by ethanol precipitation (0.1 volumes of 3 M sodium acetate followed by 2 volumes of ethanol). Pellets were washed in 70% ethanol and resuspended in water containing 10 μg/ml RNase and incubated at 37°C for 30 min. Triplicate digests were pooled and treated with with 100 U unmodified T4 polynucleotide kinase (NEB) for 30 min at 37°C to remove 3′-phosphate groups left by MNase. DNA was extracted once more with phenol:chloroform, re-precipitated with sodium acetate and propan-2-ol, washed with 80% ethanol, dried and re-suspended in TE (pH 7.5). DNA fragments were end repaired, 3′-adenylated and ligated to indexed adapters without size selection using Nextflex reagents (Newmarket Scientific, UK). Libraries were amplified with 8 cycles PCR using Kapa HiFi PCR master mix (Anachem), primers removed with GeneRead size selection protocol (QIAgen) before quantification by Bioanalyser DNA 7500 assay. Libraries were pooled, denatured, diluted to 6 nM before clustering in a single lane of a high output Illumina flowcell.  Sequencing (100nt) was undertaken on a HiSeq 2500 using TruSeq SBS v3 reagents (Illumina).