Comment[GEAAccession]	E-GEAD-319
MAGE-TAB Version	1.1
Investigation Title	Genomic localization of various factors/modificaions in HB1119 cells
Experiment Description	HB1119 cells, which express the MLL-ENL oncoprotein, was subjecteed to ChIP-seq analysis using the fanChIP/faxChIP methodology. Distributions of AF17, MOZ, BMI1, CBX8, DOT1L, MLL, TAF1C, RNAP2, H3K4me3, H3K27ac, and H3K4me2, were analyzed. To examine the contribution of wt MLL in the MLL ChIP signal, wt MLL was knocked down by sh-RNA and analyzed by ChIP-seq. fax ChIP methodology was used for H3K27me3 and H3K79me2.The ChIPed DNAs were analyzed by deep sequencing along with the input sample.
Experimental Design	binding site identification design
Experimental Factor Name	antibody
Experimental Factor Type	antibody
Person Last Name	Kanai	Yokoyama
Person First Name	Akinori	Akihiko
Person Affiliation	Tsuruoka Metabolomics Laboratory, National Cancer Center	
Person Roles	submitter	submitter
Public Release Date	2020-10-17
PubMed ID	28394257
Protocol Name	P-GEAD-231	P-GEAD-232	P-GEAD-233	P-GEAD-234	P-GEAD-235
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	HB1119 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and PS. Cell culture was performed in 5% CO2 37 degree C.The cells were  washed with PBS once and subjected to chromatin preparation.	"Chromatin fractions from HB1119 cells were prepared using the fanChIP method as previously described (Okuda et al., 2014 Nucleic Acids Res 42, 4241-56). Cells were suspended in CSK buffer and centrifuged to remove the soluble fraction. The pellet was resuspended in MNase buffer and treated with MNase at 37 degree C for 3-6 min to obtain oligonucloesomes. The MNase reaction was stopped by adding EDTA (pH 8.0) to a final concentration of 20 mM. Lysis buffer (250 mM NaCl, 20 mM sodium phosphate [pH 7.0], 30 mM sodium pyrophosphate, 5 mM EDTA, 10 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM DTT, and EDTA-free protease inhibitor cocktail) was added to increase solubility. The chromatin fraction was cleared by centrifugation and subjected to immunoprecipitation with specific antibodies and magnetic microbeads (Protein-G magnet beads [Invitrogen]). Immunoprecipitates were washed five times with washing buffer (1:1 mixture of lysis buffer and MNase buffer with 20 mM EDTA) and then eluted in elution buffer. faxChIP method is previously descrived (Okuda et al. 2017 J Clin Invest 127, 1918-1931).Cells were cross-linked by a 5-minute incu- bation with 5% formaldehyde. Glycine was added to stop the cross-link- ing reaction at a final concentration of 125 mM. After rinsing with PBS, cells were suspended in CSK buffer and centrifuged to remove the solu- ble fraction in the same manner as for the fanChIP protocol. The pellet was resuspended in MNase buffer and treated with MNase at 37 degree C for 3 to 6 minutes to obtain oligonucleosomes. The MNase reaction was stopped by adding EDTA (pH 8.0) at a final concentration of 20 mM. The chromatin was solubilized by adding SDS at a final concentration of 1% and incubated on ice for 10 minutes. The chromatin fraction was cleared by centrifugation and mixed with 2 volumes of IP dilution buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, pH 8.0, 16.7 mM Tris- HCl, pH 7.6, 167 mM NaCl, and EDTA-free protease inhibitor cocktail) and subjected to IP, as in the fanChIP protocol. faxChIP was applied for the ChIP analysis of H3K79me2 and H3K27me3."	The libralies were constructed using a TruSeq ChIP Sample Prep Kit (iIllumina) following the manufacturer's instructions.	The libraries were sequenced for 51 cycles on an Illumina HiSeq 2500 sequencer with 51-bp single-end reads.	Sequenced reads were mapped to human genome assembly hg19 using CASAVA1.8.2 or BWA 0.7.5. The BAM alignment was converted to the bBigWig coverage files using bam2wig 1.6. and wigToBigWig.
SDRF File	E-GEAD-319.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB4964
Comment[Last Update Date]	2020-10-17