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| Status |
Public on Mar 30, 2016 |
| Title |
EO041_rep2_input |
| Sample type |
SRA |
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| Source name |
L3 staged worms_input
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: L3 staged worms
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| Growth protocol |
N2 worms were grown in liquid culture (S-medium) at 25°C, fed with HB101 Escherichia coli bacteria, and harvested for ChIP-seq at the L3 stage of development (24 - 26 hrs post feeding).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flash frozen larvae were physically homogenized in a mixer mill, fixed in 1 % formaldehyde for 10 minutes, quenched with 50 mM glycin for 5 min, sheared by sonication in a Bioruptor, and the resulting chromatin was extracted. Immunoprecipitation was performed using either 5 mg a-ELT-2 (455-2A4) antibody, 5 mg a-H3K4me3 antibody (WAKO 305-34819), or mock conditions (IgG only). The anti-ELT-2 monoclonal antibodies was produce by the Southern Alberta Cancer Research Institute Antibody Services by using a purified poly-histidine tagged full-length ELT-2 protein produced in E. coli as an antigen to produce anti-ELT-2 monoclonal antibody 455-2A4 (isotype IgG1). Antibodies concentrated from hybridoma supernatants concentrated ~10-fold by pressure filtration, dialyzed extensively against PBS and then concentrated a further 2-3 fold by dialysis against PBS-50 % glycerol.
ChIP-recovered DNA and input DNA were ligated to sequencing adapters using containing an eight-base multiplexing barcode sequence. DNA libraries were sequenced on either the Hi-Seq2000 or Hi-Seq2500 Illumina sequencers with 50 rounds of single-end sequencing, 8 cycles of which were used for multiplex barcode indices.
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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 |
Quality filtering: Sequenced reads were trimmed of barcodes (FASTX-Toolkit, 11sep2008, http://hannonlab.cshl.edu/fastx_toolkit/index.html) and filtered for primer and adapter sequences using Tagdust (1.12), (Lassmann et al., 2009).
Alignment: Resulting high quality reads were aligned to the C. elegans ce10 genome using Bowtie/1.1.0 (Langmead et al., 2009). Resulting bam files were normalized to read depth and converted to bigwig format using bedtools genomecov (2.22.1), (Quinlan et al, 2010) followed by bedGraphToBigWig (UCSC Genome Browser) to create individual .bw tracks. The resulting ELT-2 and H3K4me3 ChIP-seq bigwig files were subtracted from IgG-only bigwig tracks and the resulting replicates were averaged using java-genomics-toolkit wigmath.Average (java/1.8.0_11), (http://palpant.us/java-genomics-toolkit/) to create average_track.bw files.
Peak Calling: Significant peaks were identified by MACS2 (2.0.9) using a concatenated set of ChIP-seq replicates as treatment and a concatenated set of input samples as a control (Zhang et al., 2008). Concatenated samples were downsampled to standardize the number of input reads per replicate. Peaks were filtered for a minimum threshold of -log10(q-value) ≥ 30. HOT regions (Chen et al., 2014), blacklisted regions (Araya et al., 2014) and IgG-only peaks were subtracted from the set of identified MACS2 peaks.
Genome_build: ce10
Supplementary_files_format_and_content: .bw files for individual samples; average_track.bw files merging ELT-2 or H3K4me3 ChIP-seq tracks (subtracted over IgG only tracks); bed files for significant peaks; bed files for significant peak summits.
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| Submission date |
Aug 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Erin Osborne Nishimura |
| E-mail(s) |
erinnish@colostate.edu
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| Organization name |
Colorado State University
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| Department |
Biochemistry and Molecular Biology
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| Street address |
1870 Campus Delivery, 200 W. Lake St.
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| City |
Fort Collins |
| State/province |
CO |
| ZIP/Postal code |
80523 |
| Country |
USA |
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| Platform ID |
GPL13657 |
| Series (1) |
| GSE71720 |
Defining the Roles and Regulation of the GATA-factor ELT-2 in the C. elegans Endoderm |
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| Relations |
| BioSample |
SAMN03956437 |
| SRA |
SRX1132917 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1843699_08_EO041_rep2_input.bw |
171.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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