Comment[GEAAccession]	E-GEAD-322
MAGE-TAB Version	1.1
Investigation Title	ChIP-seq analysis of HEK293T cell lines
Experiment Description	Chromaitn immunoprecipitation of HEK293T cells or its MLL-deficient derivertives (dMLLc3 and c7) was performed using fractionation-assisted native chromatin immunoprecipitation (fanChIP) method.Specific antibodies for RNAP2 non-P (8WG16), H3K18ac, H3K14ac, CCNT1, MLL, MOZ were used.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	2020-10-03
Protocol Name	P-GEAD-246	P-GEAD-247	P-GEAD-248	P-GEAD-249	P-GEAD-250
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 cells are culture 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 cells were  washed with PBS once and harvested by a cell lifter.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). 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 50 cycles on an Illumina HiSeq 2500 sequencer with 50-bp single-end reads.	Sequenced reads were mapped to human genome assembly hg19 using BWA 0.7.5. The BAM alignment was converted to the bBigWig coverage files using bam2wig 1.6 and wigToBigWig.
SDRF File	E-GEAD-322.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB8607
Comment[Last Update Date]	2020-10-03