Comment[GEAAccession]	E-GEAD-407
MAGE-TAB Version	1.1
Investigation Title	Genomic localization of RNA Polymerase II in 293T cells
Experiment Description	HEK293T cells were subjected to ChIP-seq analysis using the fanChIP/faxChIP methodologies. Distribution of  RNAP2 (non-P) was analyzed by fanChIP method. Distribution of RNAP2 (Ser5-P) was  analyzed by fax ChIP method. The ChIPed DNAs were analyzed by deep sequencing along with the input sample.
Experimental Design	binding site identification design
Experimental Factor Name	antibody	method
Experimental Factor Type	antibody	method
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	2020-12-23
Protocol Name	P-GEAD-702	P-GEAD-703	P-GEAD-704	P-GEAD-705	P-GEAD-706
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.	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. 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 degrees 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.	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 BWA 0.7.5. The BAM alignment was converted to the bBigWig coverage files using bam2wig 1.6. and wigToBigWig.
SDRF File	E-GEAD-407.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB10941
Comment[Last Update Date]	2020-12-23