Comment[GEAAccession]	E-GEAD-510
MAGE-TAB Version	1.1
Investigation Title	ChIP-Seq profiles of HIM-1, CSR-1, COH-3/4, H3K9me2, and CEC-5 at adult stage in C. elegans
Experiment Description	Chromatin fractions of C. elegans were prepared from adult populations by formaldehyde cross-linking and sonication. The worm strain was any one of N2, him-8(tm611)IV, or met-2(ok2307) set-25(n5021)III. The C. elegans chromatins and the Drosophila chromatins were mixed at a DNA ratio of 100:1. The Drosophila chromatins served as a spike-in control. Chromatin immunoprecipitations were performed with antibodies against histone H2Av in Drosophila and simultaneously with antibodies against any of HIM-1, CSR-1, COH-3/4, histone H3K9me2, CEC-5 in C. elegans, or FLAG (mock). Libraries of DNA co-immunoprecipitated with the antibodies as well as DNA of input lysates were sequenced using Illumina HiSeq sequencer in paired-end read mode. The average fragment length for these libraries was 203 bp.
Experimental Design	sex design	strain or line design	binding site identification design
Experimental Factor Name	antibody	strain
Experimental Factor Type	antibody	strain
Person Last Name	Tabara
Person First Name	Hiroaki
Person Affiliation	Genome Biology Laboratory (Kohara lab), National Institute of Genetics
Person Roles	submitter
Public Release Date	2023-02-28
Protocol Name	P-GEAD-1242	P-GEAD-1243	P-GEAD-1244	P-GEAD-1245	P-GEAD-1246	P-GEAD-1247
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	high throughput sequence alignment protocol	normalization data transformation protocol
Protocol Description	Adult populations of worms were purified using the sucrose flotation method and washed three times with M9 buffer. To weaken the cuticles, worms were agitated in 10 mM DTT and 25 mM borate-NaOH (pH 9) for 20 min. Finally, the worms washed three times with PBS.	The 50% suspension of crushed formalin-treated worms was mixed with 4 volumes of high-salt lysis buffer (0.5 M LiCl, 50 mM Tris-HCl [pH 8], 4 mM EDTA, 1% Triton-X, 0.1% deoxycholate, 0.05% sarkosyl, and 1x Complete protease inhibitors) and then sonicated to shear the chromosome DNA length to an average of 203 bp. The sonicated samples were centrifuged once. The solubilized samples (crude chromatin fraction) were filtered through membranes. The C. elegans crude chromatins and the Drosophila chromatins were mixed at a DNA ratio of 100:1. The chromatin mixtures mixed with two volumes of 8 M urea and then agitated for 15 min. The chromatin samples treated by urea were buffer-changed with Sephadex G-25 spin columns equilibrated with 1x Triton-NaCl buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8], 2 mM EDTA, 1% Triton-X, and 0.1% deoxycholate) supplemented with 0.1x Complete protease inhibitors. The urea-treated chromatin samples were used for immunoprecipitations with antibody beads.	ChIP DNA and input DNA were processed by the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645; NEB) for end-repair, 5' phosphorylation, dA-tailing, and adaptor ligation.	DNA libraries ligated to the adaptors were amplified by PCR and sequenced with the illumina HiSeq sequencer.	We prepared an integrative genome reference by combining the sequences of the C. elegans genome reference (ce10) and the Drosophila genome reference (dm6). The paired-end reads of ChIP-seq were mapped to the combined genome reference using BBmap with a set of parameters (qin=33 ambiguous=random maxindel=5 pairlen=550 maxsites2=1000 pairedonly=t). The BBmap-generated mapping data in SAM format was split into the C. elegans data and the Drosophila spike-in data, and then converted to BAM files. The peak calling on ChIP-seq data of C. elegans was performed on the basis of the MACS theory, except that the mapped reads were piled up using BEDtools. We combined the mock-ChIP data with the control lambda of the input data and then used it as the control for peak calling by MACS2.	We combined the scale-normalized mock-ChIP data with the control lambda of the input data and then used it as the control for peak calling by MACS2.
SDRF File	E-GEAD-510.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB12643
Comment[Last Update Date]	2023-02-28