SAMN06290272
External Id
Laura Landweber Lab, Biochemistry & Molecular Biophysics, Columbia University
INSDC center name
2019-06-01T00:00:00Z
INSDC first public
2019-06-01T01:21:18.990Z
INSDC last update
SRS1958987
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
SRS1958987
SRA accession
Vegetative
http://purl.obolibrary.org/obo/PO_0025223
cell type
Vegetative Oxytricha trifallax strain JRB310 was cultured at a density of 1.5 × 107 cells/L to 2.5 × 107 cells/L in Pringsheim media (0.11 mM Na2HPO4, 0.08 mM MgSO4, 0.85 mM Ca(NO3)2, 0.35 mM KCl, pH 7.0) and fed daily with Chlamydomonas reinhardtii. Cells were filtered through cheesecloth to remove debris and collected on a 10 μm Nitex mesh for subsequent experiments. To obtain native genomic DNA (containing methylation), macronuclei were purified from vegetative Oxytricha trifallax strain JRB310 using and a Nucleospin Tissue kit (Macherey-Nagel). gDNA was processed further by sucrose gradient ultracentrifugation in preparation for chromatin assembly, but not for m6dA IP-seq and MNase digest of naked DNA. Ultracentrifugation protocol: gDNA was loaded on a 15%-40% linear sucrose gradient and centrifuged in a SW40Ti rotor (Beckman Coulter) at 160,070 x g for 22.5hr at 20oC. Sucrose solutions were in 1 M NaCl, 20 mM Tris pH 7.5, 5 mM EDTA. Individual fractions from the sucrose gradient were analyzed on 0.9% agarose-TAE gels. Fractions containing high molecular weight DNA that migrated at the mobility limit were discarded as such DNA species were found to interfere with downstream chromatin assembly. All other fractions were pooled, ethanol precipitated, and adjusted to 0.5mg/mL DNA. To obtain genomic DNA lacking methylation for in vitro reconstitution, 98 chromosomes were individually PCR-amplified and purified using a MinElute PCR purification kit (QIAGEN), then mixed in equimolar ratios to obtain “mini-genome” DNA. The sample was concentrated by ethanol precipitation and adjusted to a final concentration of ~1.6 mg/ml. Xenopus histones were recombinantly expressed and purified, while Oxytricha histones were acid-extracted from purified nuclei. Xenopus or Oxytricha histones were combined in equimolar amounts in 6 M guanidine hydrochloride, 20 mM Tris pH 7.5 and the final concentration was adjusted to 1 mg/mL. The solution was dialyzed against 2 M NaCl, 10 mM Tris, 1 mM EDTA, and the octamers were purified from the tetramers and dimer species using size-exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare Life Sciences). The purity of the fractions was analyzed by SDS-PAGE and pure fractions were combined, concentrated and stored in 50% glycerol at -20°C.
growth protocol
Oxytricha trifallax
http://purl.obolibrary.org/obo/NCBITaxon_1172189
organism
Yes
presence of n6-methyladenine on dna template
Native genomic DNA
source_name
JRB310
strain
Oxy_6mA_input_replA
title