title: GSM4193057: WT1_xl_MNase; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: 0.1 g nuclei were washed once and resupended in 200 µl MNase-Buffer. Increasing amounts of MNase (8 U, 16 U, 32 U, 64 U, Sigma) were added per 0.1 g nuclei to obtain varous digestion degrees. Reactions were incubated at 30°C for 20 minutes, stopped by 15 mM EDTA and 0.5% SDS (final conc.), reversed crosslinked if required and DNA was deproteinized by Proteinase K, phenol-chloroform extracted and RNase A digested. Purified DNA was sheared to ~150 bp fragments with the Covaris S220 and concentrated using the NucleoSpin Gel and PCR Clean-up Kit (Macherey&Nagel). 0.5 – 1 µg as determined by Qubit (ThermoFisher Scientific) was used for library preparation. First, DNA was end polished (15 U T4 DNA Polymerase, 50 U T4 PNK, 5 U Klenow (NEB)) for 30 minutes at 20°C, purified with AmPure XP beads (Beckman Coulter), then subjected to A-tailing (15 U Klenow exo-(NEB)) for 30 minutes at 37°C and purified again. For adapter ligation 15 U T4 DNA Ligase (NEB) and 75 to 150 pmol NEBNext Adaptor or NEBNext Methylated Adaptor for bisulfite conversion were added. The reaction was incubated for 20 minutes at 25°C followed again by AmPure XP bead purification. Half of the end-polished and adapter ligated sample was subjected to 8 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion Polymerase (NEB). Methylated samples were bisulfite converted following the manufacturer's instructions of the Qiagen EpiTect Bisulfite Kit. 3 µl of the converted sample was subjected to 12-14 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion U Polymerase (ThermoFisher Scientific). All samples were sequenced by LaFuGa (Illumina HiSeq 1500, 50 bp paired-end mode).
attributes.tag: GEO Accession
attributes.value: GSM4193057