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| Status |
Public on Jul 27, 2016 |
| Title |
Fore wing anti Ubx ChIP pulldown rep1 |
| Sample type |
SRA |
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| Source name |
Fore Wing buds
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| Organism |
Bombyx mori |
| Characteristics |
strain: Daizo
developmental stage: larval
larval instar: IV
tissue: Fore Wing buds
chip antibody: anti-Ubx (made in-house)
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| Treatment protocol |
Nuclei were isolated from wing buds and fixed with 1% formaldehyde (in PBS) for 12 mins.The fixing was stopped by adding Glycine to a final concentration of 1.25 M for 5 minutes. Sonication of the lysed nuclei was done on Diagenode® Bioruptor XL for a total time of 15 minutes with 55 sec on /60 sec off cycle at high power.
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| Growth protocol |
Larvae were maintained at 25C and fed on Morus alba leaves.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fore and Hind wing buds (80 each) were isolated from second and third thoracic segments of the fourth instar larvae after temporarily aneasthetizing them on ice.The buds were isolated in cold PBS with Roche Protease inhibitor cocktail. The Wing buds were subjected to quick hypotonic lysis to obtain nuclei.
Chromatin Immunoprecipitation was carried out on equal volumes of wing bud Chromatin for experiment and negative control, while retaining 10% for input normalization control.The experimental pulldown was carried out using anti Bombyx N terminal Ubx antibodies generated by us. The antibodies against the 19 KDa N terminal region of Ubx protein that are specific to Bombyx Ubx were raised in Rabbit and the resultant antiserum obtained was purified against Milipore Prosep Protein A column to isolate the IgG fraction that was used for the anti Ubx ChIP pulldown. The negative control experiment was carried out using the Normal Rabbit IgG antibodies supplied with the Invitrogen Magnify Chromatin Immunoprecipitation kit, this dataset was used to eliminate non specific peaks from the anti Ubx pulldown set. Libraries for ChIP sequencing were constructed according to a modification of the Illumina ChIP Seq library protocol outlined in “Preparing Samples for ChIP Sequencing of DNA” (Part # 11257047, Rev. A, 2007). Briefly, DNA was subjected to a series of enzymatic reactions that repair frayed ends, phosphorylate the fragments, add a single nucleotide A overhang and ligate adaptors (Illumina ChIP Seq Library preparation kit). The libraries were enriched using PCR (18 cycles), post PCR cleanup was performed using Agencourt AMPURE XP beads (Beckman Coulter #A63881). The enriched PCR fragments were size selected using a 2 % Low melting agarose gel and purified using MinElute Gel Extraction Kit (QIAGEN). The prepared libraries were quantified using Nanodrop Spectrophotometer and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
FW rep 1 Experiment
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| Data processing |
The fastq files obtained were aligned to the silkworm genome ver 2.0 (SilkDB) using bowtie 0.12.7 valentine. Using the command line " ./bowtie silk_index -q ChIP_reads_file.fastq -M 1 -S -5 3 -v 3 aligned_output_file.sam -p3"
Aligned BAM files were used to call peaks with MACS ver1.4.2. Both the anti Ubx pulldown experiment or the Normal sera negative control were normalized against the Input bam files. The following parameters were used in Peak calling "macs14 -t aligned_output_experiment/negative_control-file.bam -c aligned_input_control-file.bam -g 3.11e+08 --keep-dup=2 -nNameofpeaksfile_exp_vs_input"
The peaks common to Negative and anti Ubx datasets were deleted. An FDR cutoff of 15% was used to select the final set of peaks.
The common peaks between replicate 1 and 2 were selected through bedtools intersect tool with a minimum intersect of 1 nucleotide overlap.
Genes were allocated to a peak using Galaxy tool "fetch both up and downstream " and also intersecting the peak file with Bombyx gtf annotation file (SilkDB v2.0) to obtain all genes corresponding to a peak spanning 10Kb up and downstream.
Genome_build: SilkDB ver 2.0 silkworm genome. (ftp://silkdb.org/pub/current/Genome/silkworm_genome_v2.0.fa)
Supplementary_files_format_and_content: Excel sheets with peak locations (chr (scaffold), start, end, length, summit, tags, MACS_peak_#, -10*log10(pvalue), fold enrichment, FDR(%)).
Supplementary_files_format_and_content: Bed file header- chr, start, end, MACS_peak_#, -10*log10(pvalue), fold enrichment, FDR(%)
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| Submission date |
Aug 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Shreeharsha Tarikere |
| E-mail(s) |
harsha.tts@gmail.com
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| Organization name |
Harvard University
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| Department |
Organismic and Evolutionary Biology
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| Lab |
Dr.Cassandra G. Extavour
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| Street address |
16 Divinity Avenue, Biolabs
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL9151 |
| Series (2) |
| GSE71990 |
Identification of genome wide targets of the hox protein Ultrabithorax (Ubx) in larval wing buds of Bombyx mori (Daizo) |
| GSE71992 |
A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species. |
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| Relations |
| BioSample |
SAMN03983386 |
| SRA |
SRX1143505 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1849360_Hind_wing_rep2_anti_Ubx_ChIP.xls.gz |
648.2 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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