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Status |
Public on Jan 12, 2020 |
Title |
ChIP_H2AK118Ub_E1416_rep2 |
Sample type |
SRA |
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Source name |
ChIP_H2AK118Ub_E1416
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Organism |
Drosophila melanogaster |
Characteristics |
strain: W1118 Oregon-R develpmental stage: Whole 14-16h embryo tissue: whole embryo chip antibody: Cell Signaling Technology; Cat. N. 8240S (D27C4)
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Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP from ED or whole 14-16h embryos were performed in duplicate as previously described in Loubiere et al., 2016. Briefly, ~200 ED from wandering larvae were used per replicate for PTM ChIP-Seq and ~500 for PSC and SU(Z)12 ChIP-Seq. For each replicate of PTM ChIP-Seq from 14-16h embryos, 2h collections were performed at 25°C with about 500 flies. When necessary, several batches of dissection/collection were snap-frozen in liquid nitrogen and stored at -80°C in order to collect enough material. Samples were then transferred into a tight Tenbrock and cross-linked for 15min at RT using 1.8% formaldehyde and continuous homogenization before being lysed on ice for 2h with 1% SDS Lysis Buffer 2 (see Loubiere et al., 2016). Chromatin was sonicated using a Bioruptor (Diagenode) for 18 cycles (settings 30s ON, 30s OFF, high power) and the quality of the sonication was checked on a 1.5% agarose gel (~300bp fragments). Then, samples were pre-cleared O/N using 15µL of protein A Dynabeads (ThermoFisher Scientific, 10001D) at 4°C and an aliquot was kept apart to constitute the INPUT. Antibodies were added (see Supplementary Table 7) 4h prior to the addition of 30µL of protein A dynabeads and O/N incubation at 4°C. Finally, beads were washed, and the precipitated chromatin was eluted before O/N decrosslinking at 65°C, Proteinase K treatment, Phenol/Chloroform purification and O/N ethanol precipitation. Libraries were prepared using the TruSeq ChIP Sample Preparation kit from Illumina, following manufacturers’ instructions.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
After initial quality checks using fastqc, paired-end and single-end .fastq files were aligned to the dm6 version of the Drosophila genome using bowtie 2 with default parameters (v2.1.0). Reads with low mapping quality (mapq<30) were discarded using samtools. Peak calling was performed on each replicate separately and on the merged replicates using MACS2 (v2.1.1.20160309) with the following parameters: -g dm --nomodel --keep-dup 1 --extsize 200. For the visualization and the quantification of ChIP-Seq tracks, RPKM-normalized bigwig binary files were generated using the bamCoverage function from Deeptools2 (v2.5.1) with the following parameters: -of=bigwig --samFlagExclude 128 --ignoreDuplicates -e 200 –normalizeUsingRPKM. Finally, replicates were merged using the bigWigMerge and bedGraphToBigWig tools from UCSC with default parameters. Supplementary_files_format_and_content: bw files correpond to the merged (2 replicates) RPKM-normalized bigwig binary files (10bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software. NarrowPeak files correpond to the peaks that were called using the MACS2 software on separated replicates and on the merged replicates (10 columns: chrom, chromStart, chromEnd, name, score, strand, signalValue, pValue, qValue, peak). Summit file contains the summits of the peaks called from the merged replicates. Compressed HiC tracks; Binary files of intrachromosomal pairwise interaction probabilities (score) computed using the "shaman" package in R. Ouput file of differential expression analysis using the DESeq2 R package. 8 Columns: FBgn (FlyBase Gene ID); Symbol; baseMean; log2FoldChange; lfcSE; stat; pvalue; padj. Genome_build: dm6
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Submission date |
Feb 24, 2019 |
Last update date |
Jan 12, 2020 |
Contact name |
Vincent Loubiere |
E-mail(s) |
vincent.loubiere@imp.ac.at
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Phone |
+33663182164
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Organization name |
UMR9002; CNRS-UM
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Department |
Institute of Human Genetics
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Lab |
Chromatin and Cellular Biology
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Street address |
141 rue de la Cardonille
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City |
Montpellier |
State/province |
FRANCE |
ZIP/Postal code |
34090 |
Country |
France |
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Platform ID |
GPL13304 |
Series (1) |
GSE126985 |
PRC1 facilitates stage-specific enhancer-promoter contacts during Drosophila development |
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Relations |
BioSample |
SAMN10994982 |
SRA |
SRX5416190 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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