Comment[GEAAccession] E-GEAD-323 MAGE-TAB Version 1.1 Investigation Title ChIP-seq analysis of the HEK293T cell line transiently expressing various FLAG-tagged proteins Experiment Description Chromaitn immunoprecipitation of HEK293T cells transiently expressing FLAG-tagged MOZ5', MLL5', and MTM was performed using fractionation-assisted native chromatin immunoprecipitation method along with the mock control. Specific anti-FLAG antibody (M2) was used for IP. The ChIPed DNAs were analyzed by deep sequencing. Experimental Design binding site identification design Experimental Factor Name FLAG-tagged protein Experimental Factor Type FLAG-tagged protein 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 2020-10-04 Protocol Name P-GEAD-251 P-GEAD-252 P-GEAD-253 P-GEAD-254 P-GEAD-255 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 HEK293T cells were transfected with various constructs using lipofectamine 2000. Medium was changed once 6-8h after trasnfection. 24h after transfection, the cells were washed with PBS and harvested by a cell lifter.The cell suspensions were washed with PBS once and subjected to chromatin preparation.\" "Chromatin fractions from HEK293T derivatives 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 anti-FLAG M2 antibody and magnetic microbeads (Protein-G magnet beads [Invitrogen]) and 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-323.sdrf.txt Comment[AEExperimentType] ChIP-seq Comment[BioProject] PRJDB8608 Comment[Last Update Date] 2020-10-04