title: GSM4193095: WT4_xl_CpG_2U_240min_refill_at_180min_NP-seq; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Nuclei pellets were washed in methylation buffer (20 mM HEPES-NaOH pH 7.5, 70 mM NaCl, 0.25 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.5% (v/v) glycerol, 1 mM DTT, 0.25 mM PMSF (Darst et al. 2012)). Per reaction, nuclei from ca. 0.1 g wet cell pellet were resuspended in 800 µl methylation buffer containing 640 µM freshly added S-adenosylmethionine (SAM). 200 U M.SssI or M.CviPI (both NEB) and 10 mM DTT were freshly added. Methylation reactions were dialyzed in Slide-A-Lyzer MINI Dialysis Devices 10 K MWCO (Thermo Fisher Scientific) against 15 ml methylation buffer with 200 µM freshly added SAM at 25°C for M.SssI or 37°C for M.CviPI. 0.5 - 1 µg fully assembled pUC19-601-25mer plasmid (pFMP233) (Lowary and Widom 1998; Lieleg et al. 2015a) and SGD assembled Escherichia coli gDNA or SGD assembled E. coli plasmid library (limited Sau3A fragments of E. coli gDNA ligated into pJET 1.2 plasmid (Thermo Fisher Scientific)) was spiked-in prior to methylase addition. E. coli spike-in results were not further pursued due to low coverage. Reactions were stopped by 0.5% SDS (final conc.), reversed crosslinked if required and DNA was deproteinized by Proteinase K, phenol-chloroform extracted and RNase A digested. Oxford Nanopore library construction and sequencing  For Nanopore sequencing, 1 µg of purified DNA was subjected to 1D native barcoding (Oxford Nanopore, SQK-LSK109). To get up to 1 Gbases coverage per sample, up to five samples were loaded to a MinION flowcell (R9.4.1).
attributes.tag: GEO Accession
attributes.value: GSM4193095