Comment[ArrayExpressAccession]	E-MTAB-1685
MAGE-TAB Version	1.1
Investigation Title	Profiling of H3.3
Comment[Submitted Name]	Profiling of H3.3
Experiment Description	Histone variants can effect nucleosome stability or affect histone of DNA modifications. H3.3 is a major H3 histone variant that is incorporated into chromatin outside of S-phase in various eukaryotes. In animals, H3.3 is associated with active transcription and possibly maintenance of transcriptional memory. Plant H3.3, which evolved independently of animal H3.3, is much less well understood. We performed ChIP-chip using chromatin from rosette leaves of 35S:H3.3-YFP plants.
Experimental Design	binding site identification design	replicate_design
Comment[AEExperimentType]	ChIP-chip by array
Comment[AEExperimentDisplayName]	ChIP-chip by array using chromatin from rosette leaves of 35S:H3.3-YFP Arabidopsis thaliana plants in order to profile H3.3, a major H3 histone variant
Experimental Factor Name	immunoprecipitate
Experimental Factor Type	immunoprecipitate
Quality Control Type	biological replicate
Quality Control Term Source REF	EFO
Public Release Date	2014-03-10
Person Last Name	Hennig
Person First Name	Lars
Person Mid Initials
Person Email	lars.hennig@slu.se
Person Phone
Person Address
Person Affiliation	Swedish University of Agricultural Sciences
Person Roles	submitter
PubMed ID	24708891
Publication Author List	Shu H, Nakamura M, Siretskiy A, Borghi L, Moraes I, Wildhaber T, Gruissem W, Hennig L.
Publication Title	Arabidopsis replacement histone variant H3.3 occupies promoters of regulated genes.
Publication Status
Protocol Name	P-MTAB-32990	P-MTAB-32991	P-MTAB-32992	P-MTAB-32993
Protocol Type	growth protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	ChIP
Protocol Description	Experimental plants were grown on soil at 21C in dark (16 h) and 20C in light (8 h). Plant age was recorded as days after seeds were transferred to growth chamber (days after induction of germination). Leaves (leaf no. 6 from ~5 plants per sample) were harvested after 35 d at zeitgeber time 7 (i.e. 7 h after start of the photoperiod), and frozen in liquid nitrogen. Note that cell division and expansion had ceased in the harvested leaves.	DNA was extracted using phenol-chloroform extraction and ethanol/salt precipitation	DNA amplification was performed using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma) followed by purification using MinElute PCR Purification kit (Qiagen, Germany). Q-PCR was performed for 6 genomic fragments before and after amplification to control for amplification bias. Amplified ChIP DNA was fragmented, labelled and hybridized to Affymetrix AGRONOMICS1 Arabidopsis tiling arrays.	Native Chromatin Immunoprecipitation (ChIP) was performed as described [Bernatavichute, 2008 #7690] with minor modifications. Crude nuclei extracts were produced by treating 100 mg of ground frozen plant material in Nuclei Extraction Buffer (NEB; 20 mM PIPES-KOH pH 7.6, 1 M hexylene glycol, 10 mM MgCl2, 0.1 mM EGTA, 15 mM NaCl, 60 mM KCl, 0.5% Triton-X, 5 mM mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche)) for 15min at 4C. The homogenate was filtered through Miracloth (Calbiochem, Germany), and a pellet was collected by centrifugation for 10 min at 1500xg at 4C. Isolated nuclei were washed once in MNase buffer (50 mM Tris-HCl pH 8, 10 mM NaCl, 5 mM CaCl2, and EDTA-free protease inhibitor cocktail (Roche)), treated with 1.3 ul of RNase A, 30 ug/ul (Sigma-Aldrich, Missouri) and used for Micrococcal Nuclease (New England BioLabs, NEB, Massachusetts) digestion for 4 minutes (final concentration 0.2 U/ul) in MNase buffer. The reaction was stopped with 10 mM EDTA. The supernatant after a centrifugation was collected as phase 1 chromatin preparation, and the supernatant from a second centrifugation after a treatment of the pellet with buffer S2 (1 mM Tris-HCl pH 8, 0.2 mM EDTA, and EDTA-free protease inhibitor cocktail (Roche)) for 30 min was collected as phase 2 chromatin preparation. The two phases of chromatin preparations were combined and the NaCl concentration was adjusted to 50 mM. The majority of the chromatin was of mononucleosome size (data not shown). Histone H1 was depleted by incubating the chromatin preparation with Sephadex C25-CM resin (Pharmacia, Sweden) for 1h at 4C (Thorne et al, 2004). The Triton-X concentration in the mononucleosomal chromatin was brought up to 0.1% followed by preclearing using non-immune rabbit IgG (see below) and Dynabeads Protein A (Invitrogen). One tenth of the precleared mononucleosomal chromatin was kept as input control, and ¼ was used for each immunoprecipitation with 2.5 ug antibody (MAB3422, monoclonal anti-histone antibody, Upstate/Millipore, Massachusetts; #A11122, polyclonal anti-GFP antibody, can also recognize YFP, Invitrogen; #I5006 non-immune rabbit IgG, reconstituted in H2O, Sigma-Aldrich) and collected with Dynabeads Protein A (Invitrogen). After washing, beads were re-suspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and DNA was extracted using phenol-chloroform extraction and ethanol/salt precipitation. ChIP was performed in biological triplicates.
Protocol Term Source REF	EFO	EFO	EFO	EFO
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-1685.sdrf.txt
Publication DOI	10.1186/gb-2014-15-4-r62