Comment[GEAAccession]	E-GEAD-585
MAGE-TAB Version	1.1
Investigation Title	Genomic localization of ENL and DOT1L in HEK293T cells deficient for MOZ
Experiment Description	To elucidate the role of MOZ in the genomic localization of ENL and DOT1L, we generated HEK293T cells  lacking the both alleles of MOZ by CRISPR-cas9 technologies. HEK293T cells deficient for MOZ were analyzed using ChIP-seq technologies with anti-ENL and DOT1L antibodies. For comparison, the parental HEK293T cells were also anlyzed along side.
Experimental Design	cell type comparison design	binding site identification design
Experimental Factor Name	genetic_modification	antibody
Experimental Factor Type	genetic_modification	antibody
Person Last Name	Yokoyama	Sequence
Person First Name	Akihiko	Kashiwa
Person Affiliation	National Cancer Center Tsuruoka Metabolomics Laboratory	
Person Roles	submitter	submitter
Public Release Date	2023-03-07
Protocol Name	P-GEAD-1667	P-GEAD-1668	P-GEAD-1669	P-GEAD-1670	P-GEAD-1671
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	HEK293T cell lines (parental and dMOZ) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (PS).Cell culture was performed in 5% CO2 37 degree C.  The cell suspensions were washed with PBS once and subjected to chromatin preparation.	Chromatin fractions from HEK293T cells were prepared using the fanChIP method as previously described (Okuda et al., 2014 Nucleic Acids Res 42, 4241-56, Miyamoto and Yokoyama 2021 STAR protocols 2, 100404 ). 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	The libralies were constructed using a TruSeq ChIP Sample Prep Kit (iIllumina) following the manufacturer's instructions.	The libraries were sequenced for 100 cycles on an Illumina Novaseq 6000 sequencer with 150-bp paired-end reads.	Sequenced reads were mapped to human genome assembly hg19 using Bowtie 2 v2.3.5.1. The BAM alignment was converted to the bBigWig coverage files using bam2wig 1.6. and wigToBigWig.
SDRF File	E-GEAD-585.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB14995
Comment[Last Update Date]	2023-03-07