title: GSM4193221: in_vitro_chromatin_low_dens_GpC_20U_30min_MgCl2; 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. 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: GSM4193221