biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsDCC modENCODEChIP-chip by tiling arrayCaenorhabditis elegansCaenorhabditis eleganshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-37511modENCODE_submission_3558 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. 126 strategically selected targets include key histone modifications, histone variants, 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, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions. 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-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: L4 Larva; Genotype: wild type; Sex: mixed Male and Hermaphrodite population; NUMBER OF REPLICATES: 1; EXPERIMENTAL FACTORS: Developmental Stage L4 Larva; temp (temperature) 20 degree celsius; Antibody HK00001 H3K36me3:13C9 (target is H3K36me3); Strain N2biostudies-arrayexpressHybridization - ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.Nucleic Acid Extraction - Worm_extraction_vVR1.Embryos or worms were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was sonicated 15 times on ice at the following settings: 10 sec; 40% power output. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4?C and taking the supernatant. Protein concentration was determined, and extracts were aliquoted and stored at -80?C. 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_LM-PCR_Amplification_for_ChIP-chip_vSS2. ChIP DNA was amplified with a modified ligation mediated PCR (LM-PCR) protocol derived from FlyChip protocol version 1.2 from R. Auburn: http://www.flychip.org.uk/protocols/chip/lm_pcr.php. For a detailed protocol see http://www.modencode.org/.Labeling - ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42°C.Growth Protocol - Worm_growth_and_harvest_vVR1. Worms were grown to the desired stage and cross-linked in M9 + 1.85% formaldehyde for 30 minutes at room temperature. Embryos or worms were then washed twice with M9 Buffer, once with 100mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C.MIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsThea EgelhoferSusan StromeDCC modENCODEImage Adquisition - ChIP-chip_scanning_nimblegen_v1. Array scanning and raw data extraction were performed at Roche NimbleGen, according to the protocol described in chapter 5 and 6 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, array signal was scanned by using a GenePix 4000B Scanner with associated software and saved as .tif files of the 532nm and 635nm images individually. Raw signal intensities of the images were extracted and saved as .pair files by using NimbleScan software according to the NimbleScan v2.4 User?s Guide.Feature Extraction - ChIP-chip normalization standard MA2C:JL:2 protocol. ChIP-chip_normalization_standard_MA2C_v2. First, all the IP and INPUT log ratio values are read from the pairdata file. Secondly, we build GC bins for INPUT and IP based on the GC counts for every probe sequence, which means that the INPUT or IP values for any probes which have the same GC counts will be put together. After that, for each GC bin, we calculate the mean for IP and INPUT data, and the covariance between this two channels. By default, the robust mean variance method is applied, which generalizes Tukey's theory of bi-weight estimation where the constant C is set to 2. At last, we adjust the log ratio values for each probe by using the mean and covariance values for their corresponding GC bins, then these values are further normalized by their mean and standard derivation. In a single experiment, median within the sliding window defined by 2x bandwidth parameter is assigned as MA2C score at the center of each probe. In case of replicates, when we calculate the MA2Cscore afterwards, we take the median as the score from all the replicates for all the probes within the sliding window defined by 2x bandwidth parameter. Processed data are obtained using following parameters: genome version is WS190falseStrome HK00001_H3K36ME3_13C9_N2_L4_s6modENCODE_submission_3558 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. 126 strategically selected targets include key histone modifications, histone variants, 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, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions. 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-chip. BIOLOGICAL SOURCE: Strain: N2; Developmental Stage: L4 Larva; Genotype: wild type; Sex: mixed Male and Hermaphrodite population; NUMBER OF REPLICATES: 1; EXPERIMENTAL FACTORS: Developmental Stage L4 Larva; temp (temperature) 20 degree celsius; Antibody HK00001 H3K36me3:13C9 (target is H3K36me3); Strain N22012-04-22T00:00:00Z2023-08-23T12:08:16.618Z2021-10-01T18:08:17ZE-GEOD-37511GSE37511EFO_0002762