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| Status |
Public on Oct 15, 2015 |
| Title |
nanosGAL4_dsCG9754_rep1_H3K9me3ChIPseq |
| Sample type |
SRA |
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| Source name |
adult fly ovaries
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| Organism |
Drosophila melanogaster |
| Characteristics |
age: 3-5 days old
chip antibody: H3K9me3 (abcam, ab8898)
tissue: adult fly ovaries
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| Treatment protocol |
Flies were fed with yeast paste. Germline specific knockdowns were done as described (Czech et al., 2013 Mol Cell). Dissected ovaries were crosslinked with 2% formaldhyde for 10min at room temperature and quenched with Glycine for 10min before lysis.
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| Growth protocol |
All fly stocks were maintained at 25°C on standard diet.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody against H3K9me3 (ab8898).
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, Taq DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Taq DNA polymerase and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15-18 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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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 |
Any identifiable sequencing barcodes (at the 5' end of reads) and adaptors (Illumina TruSeq adaptors, present at the 3' end of the reads) were clipped using the fastx toolkit version 0.0.13.2. For ChIP-seq data, sequences <20 nt in length after clipping were discarded. A set of canonical transposon sequences for D. melanogaster (version 9.42) was obtained from FlyBase (http://flybase.org). The dm3 release of the D. melanogaster genome was obtained from the University of California at Santa Cruz genome database. To this genome database, we also added the X-TAS heterochromatic sequences, which represent GenBank entry L03284 and comprise X-telomeric TAS repeats from the OregonR strain. For genomic annotations, we used the D. melanogaster release 5.57 gene annotation file in gff format from FlyBase (http://flybase.org). The H3K9me3 ChIP-seq data were mapped first to the transposon database using the Bowtie2 software package (version 2.3.0). Due to the repetitive nature of transposon sequences, reads were allowed to map to up to 10 different canonical transposons, with read counts distributed evenly among all potential matches. All reads that failed to align to the transposon database were then mapped to the genome using the same criteria. The sum of the number of reads mapped to the genome and transposon database is used as the actual “library size”, and used later for normalization. To obtain the genome-wide enrichment of H3K9me3, we re-mapped all reads to the genome with one mismatch and allowing uniquely mapped reads only, and removed presumptive PCR duplicates with the same mapped coordinates.
We used the SICER software package (version 1.1) to call significantly enriched peaks at a false discovery rate (FDR) of 0.05 relative to the input samples. For the SICER peak-calling program, we additionally set the parameters such that reads were binned in windows of 500 bp, aggregating all peaks within a distance of 500 bp.
For differential enrichment analysis, we combined genomic peaks identified above and transposons with 2 fold changes against the input and counts >100, and used the DESeq package, with parameters advisable for data sets with few replicates (2) (fit, “local’’; method, “pooled’’; and sharing Mode, ‘‘fit-only’’), accepting only changes greater than twofold, with adjusted p-values < 0.05.
Genome_build: dm3/2006
Supplementary_files_format_and_content: In sheet "HetA_RPM", "TART_RPM", and "Firefly_transgene_locus_RPM", normalized read counts of H3K9me3 ChIP mapping to each position of the indicated genes or transposon consensus. In sheet "Untethered_vs_tethered_RPM", normalized read counts of H3K9me3 ChIP mapping to the indicated genes. In sheet "het_vs_mut_fold" and "nanosGAL4_control_vs_gene_fold", fold changes (log2) for each transposon consensus were shown deviding H3K9me3 ChIP reads from control by that from the indicated experiments (Klenov et al., NAR, 2013).
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| Submission date |
Jul 27, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Yang Yu |
| E-mail(s) |
[email protected]
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| Organization name |
Cold Spring Harbor Laboratory
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| Lab |
Hannon Lab
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| Street address |
One Bungtown Road
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE71368 |
Panoramix enforces piRNA-dependent co-transcriptional silencing (ChIP-Seq) |
| GSE71374 |
Panoramix enforces piRNA-dependent co-transcriptional silencing |
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| Relations |
| BioSample |
SAMN03939054 |
| SRA |
SRX1120692 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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