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| Status |
Public on Mar 11, 2017 |
| Title |
LET-418_YA_rep2 |
| Sample type |
SRA |
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| Source name |
young adults, LET-418 ChIP
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain/background: N2
tissue: whole body
age: young adults
chip antibody: LET-418 (Strategic Diagnostics Inc., Q3861)
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| Growth protocol |
About 2-7 million adults grown in liquid culture at 20C were bleached and then hatched in M9 for 24 hrs. Hatched L1 larvae were inoculated into liquid culture (S-basal and HB101 bacteria) at a density of less than 1 million per 100ml. After 60hrs at 20C, young adults were harvested, washed in M9 buffer, cleaned by sucrose flotation and collected by freezing in liquid nitrogen. An aliquot was stained for DAPI to confirm the stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen worm pellets were ground, crosslinked for 10 minutes in 1.5 mM EGS in FA buffer, then formaldehyde added to 1% and cross-linking continued for an additional 10 minutes (protein factors), or fixed 10 minutes in 1% formaldehyde (histone modifications). Fixative was quenched with 125 mM glycine and cross-linked tissue washed 2X with PBS + protease inhibitors, then resuspended in FA buffer and subjected to sonication in a Bioruptor or Bioruptor pico to an average DNA size of 200bp. Extracts were spun down and the soluble fraction used for ChIP. For ChIPs of protein factors, 1mg of ChIP extract was incubated with 5ug antibody; for histone modifications, 500ug ChIP extract was incubated with 2ug of antibody. After overnight incubation with rotation at 4C, 40ul of equilibrated magnetic beads (coupled to protein A or G, depending on antibody) were added and incubated for 2 hrs at room temperature with rotation. Washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer were performed and DNA was eluted in elution buffer (1% SDS in TE with 250 mM NaCl) two times with 57 ul volume each, at 65°C. Samples were treated with RNase, proteinase K and then crosslinks were reversed overnight at 65°C. DNA was purified on Qiagen PCR purification columns and used for ChIP library preparation.
ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads. Illumina TruSeq adaptors and barcodes were used. Samples were sequenced on a HiSeq1500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Libraries were sequenced using Illumina HiSeq and de-multiplexed FASTQ files were obtained from Illumina BaseSpace.
Reads were aligned to the WS220/ce10 assembly of the C. elegans genome using BWA v. 0.7.7 with default settings (BWA-backtrack algorithm).
The SAMtools v. 0.1.19 ‘view’ utility was used to convert the alignments to BAM format.
To be able to investigate binding and expression at repetitive elements, we used all aligned reads (mapq0) to generate pileup and normalised tracks.
ChIP-seq data were normalized using summed input and the BEADS algorithm, omitting the mappability correction step (Cheung et al., 2011).
ChIP-seq signals were exported to BigWiggle tracks at 1bp native resolution.
Genome_build: WS220/ce10
Supplementary_files_format_and_content: "linear" scores in bigWig tracks represent BEADS values (signal relative to input); files were generated using BEADS normalization pipeline in R.
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| Submission date |
Sep 30, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie Ahringer |
| E-mail(s) |
ja219@cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
The Gurdon Institute
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL18730 |
| Series (2) |
| GSE87522 |
A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress [ChIP-seq] |
| GSE87524 |
A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress |
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| Relations |
| BioSample |
SAMN05855005 |
| SRA |
SRX2202782 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2333104_LET418_YA_ChIPseq_BEADSmapq0_rep2.bw |
207.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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