{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["DCC modENCODE"],"study_type":["ChIP-seq"],"organism":["Caenorhabditis elegans"],"species":["Caenorhabditis elegans"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-GEOD-50332"],"description":["modENCODE_submission_6329 This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. Our 126 strategically selected targets include RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents. We will integrate information generated with existing knowledge on the biology of the targets and perform ChIP-seq analysis on mutant and RNAi extracts lacking selected target proteins. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: Early Embryo; Genotype: wild type; Sex: mixed Male and Hermaphrodite population;  EXPERIMENTAL FACTORS: Developmental Stage Early Embryo; temp (temperature) 20 degree celsius; Strain N2; Antibody T26A5.5 SDQ3867 (target is T26A5.5)"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Worm_embryo_extraction_vSS6. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (40-48 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/. DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel.","Library Construction - Worm_embryo_extraction_vSS6. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (40-48 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP. Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/. Worm_ChIP-seq_ DNA_Library_Prep_vSS4. ChIP and 1 ng of input are blunted (T4 polymerase), an A-overhang is added (Klenow), DNA is ligated (concentrated T4 ligase) to annealed adaptors, amplified by PCR, and size-selected to the 200-400 bp range using a Pippin prep.","Growth Protocol - Worm_embryo_growth_and_harvest_vSS8. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were then washed once with PBS. Pellets were flash frozen by adding drop-wise directly to liquid nitrogen, and stored at -80C until ready for use. For a detailed protocol see http://www.modencode.org/."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"pubmed_authors":["Andreas Rechtsteiner","Susan Strome","Thea Egelhofer","DCC modENCODE"],"additional_accession":[]},"is_claimable":false,"name":"seq-SDQ3838_T26A5.5_N2_Eemb","description":"modENCODE_submission_6329 This submission comes from a modENCODE project of Jason Lieb. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. Our 126 strategically selected targets include RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents. We will integrate information generated with existing knowledge on the biology of the targets and perform ChIP-seq analysis on mutant and RNAi extracts lacking selected target proteins. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: Early Embryo; Genotype: wild type; Sex: mixed Male and Hermaphrodite population;  EXPERIMENTAL FACTORS: Developmental Stage Early Embryo; temp (temperature) 20 degree celsius; Strain N2; Antibody T26A5.5 SDQ3867 (target is T26A5.5)","dates":{"release":"2013-08-28T00:00:00Z","modification":"2023-09-06T06:26:41.762Z","creation":"2021-10-06T15:54:40Z"},"accession":"E-GEOD-50332","cross_references":{"GEO":["GSE50332"],"EFO":["EFO_0002692"]}}