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| Status |
Public on Sep 05, 2019 |
| Title |
rex-14rex-47rex-8insertion_SDC-3_ChIP-seq |
| Sample type |
SRA |
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| Source name |
mixed-stage embryos
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain name: TY5867
genotype: yIs196(rex-14) yIs190(rex-47) yIs192(rex-8) I; rex-14(y738) rex-47(y671) rex-8(y737) X
antibody: anti-SDC-3
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| Treatment protocol |
Embryos were washed once in 2% formaldehyde in M9 buffer and then fixed for 30 min with gentle rocking in 50 mL 2% formaldehyde in M9 buffer. Embryos were washed in 10mM Tris-HCl (pH 7.5) and then in FA buffer (150 mM NaCl, 50 mM HEPES-KOH pH 7.6, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and resuspended in FA buffer with protease inhibitors (5 mM DTT, protease inhibitor cocktail, 1 mM PMSF) to a total volume of 1 mL.
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| Growth protocol |
Worms were grown on RNAi plates with HT115 bacteria carrying the empty plasmid vector pL4440. Mixed stage embryos were isolated by bleaching and frozen in liquid nitrogen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryos were ground with 50 strokes in a 2 mL dounce homogenizer. Sarkosyl was added to a final concentration of 0.1% and chromatin was sheared in an S2 Covaris with duty cycle 20%, intensity 8, and 200 cycles/burst for 30 cycles of 60 sec with 45 sec of rest for a total time of 52 min. The extract was centrifuged at maximum speed for 15 min at 4° C. Supernatant containing approximately 2 mg total protein was incubated with 6.6 μg rabbit polyclonal anti-DPY-27 (rb699) (Chuang et al., 1994), rat polyclonal anti-SDC-3 (PEM4A) (Crane et al., 2015), or random IgG antibodies overnight at 4° C in a volume of at least 500 μl. 50 μl of Protein A Dynabeads (ThermoFisher Scientific, 10002D) were washed in FA buffer three times, added to the immunoprecipitation, and mixed at 4° C for at least 2 hr. Beads were then washed and DNA eluted as in (Kruesi et al., 2013).
We performed end repair, A-tailing, ligation of NEXTflex DNA Barcodes, and amplification as in Kruesi et al 2013.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Adapters were trimmed using cutadapt version 1.2.1 (Martin, 2011), and reads were then aligned to the ce11 genome using bowtie2 version 2.3.0 (Langmead and Salzberg, 2012) with default settings. For comparisons involving strains with rex site insertions, the reference genome was modified to incorporate the rex insertions.
Reads were sorted using SAMtools version 1.3.1 (Li et al., 2009) and read coverage in bigwig files was calculated by normalizing the read number in each 50 bp bin to the total read number using the bamCoverage function in deepTools version 2.5.0.1 (Ramírez et al., 2016) with the “normalizeUsingRPKM” option. Analysis of data from wild-type and 8rexΔ strains was performed with two combined biological replicates, and one replicate was used for data from TY5872 8rexΔ plus rex-32 & rex-8 and TY5867 3rexΔ plus rex-14, rex-8 & rex-47 strains.
Genome_build: ce11
Supplementary_files_format_and_content: bigWig files contain reads normalized by the total read number (RPKM) in 50 bp bins
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| Submission date |
Mar 19, 2019 |
| Last update date |
Sep 07, 2019 |
| Contact name |
Elphege P Nora |
| E-mail(s) |
elphege.nora@ucsf.edu
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| Organization name |
UCSF
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| Department |
CVRI
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| Lab |
Nora
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| Street address |
555 Mission Bay Blvd S
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL22765 |
| Series (2) |
| GSE128564 |
X chromosome domain architecture regulates Caenorhabditis elegans lifespan but not dosage compensation [ChIP-seq] |
| GSE128568 |
X chromosome domain architecture regulates Caenorhabditis elegans lifespan but not dosage compensation |
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| Relations |
| BioSample |
SAMN11173227 |
| SRA |
SRX5545246 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3680205_rex-14rex-47rex-8insertion_SDC-3.bw |
10.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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