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| Status |
Public on Feb 27, 2009 |
| Title |
1hr_Recovery_4H8 |
| Sample type |
SRA |
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| Source name |
1hr recovery from 12hr L1 arrest
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| Organism |
Caenorhabditis elegans |
| Characteristics |
wild-type strain N2
variable: condition: 1 hr recovery from 12 hr L1 arrest; antibody: 4H8 (ab5046)
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| Treatment protocol |
Larvae were cross-linked for 30 min in 2% Formaldehyde (Polysciences, Inc.) in a shaking incubator at 20°C. Formaldehyde was quenched by adding 0.1 volume of 1 M Tris pH 7.4. Larvae were washed in ice-cold S-basal plus 125 mM Glycine, followed by ice-cold S-basal, and they were spun down to a small volume and frozen.
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| Growth protocol |
Nematode cultures were maintained, passaged, and collected at 20°C. A fresh thaw of C. elegans strain N2 from the Sternberg laboratory strain collection was used for microarray analysis; this N2 strain was obtained from the Caenorhabditis Genetics Center in 1987, expanded and frozen. The worms were thawed and expanded on agar plates with E. coli OP50 as food {Lewis, 1995 #5}. Populations of worms were synchronized in liquid culture for two generations prior to staging for ChIP analysis. A starved 5 cm plate was used to inoculate a 10 ml liquid culture (S-complete plus 40 mg/ml E. coli HB101), and the liquid culture was incubated for 3 d and then bleached to produce a clean preparation of embryos {Lewis, 1995 #5}. One million embryos were suspended in 100 ml of S-complete plus 40 mg/ml HB101 and they were cultured for 70 hr at 180 rpm. Cultures were bleached resulting in approximately 10 embryos per worm bleached. Bleached embryos were examined to ensure that they were clean and healthy and resuspended in S-complete at 10 embryos/microliter with no HB101. They were cultured at 180 rpm for either 18 hr (for 6 hr L1 arrest) or 24 hr (for 12 hr L1 arrest) allowing them to hatch and enter L1 arrest, and then they were crosslinked. Alternatively, after 24 hr of culture, 25 mg/ml HB101 was added to recovery the worms from arrest and they were cross-linked after 1 hr.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In preparation for sonication, larvae were thawed on ice, washed once in Farnham Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40, Roche Protease Inhibitor Cocktail, and Thermo Scientific HALT Phosphatase Inhibitor Cocktail), suspended in 2 ml of Farnham Lysis Buffer, and incubated on ice for 15 min with occasional mixing. Larvae were pelleted, washed once with RIPA Buffer (1xPBS, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, Roche Protease Inhibitor Cocktail, and Thermo Scientific HALT Phosphatase Inhibitor Cocktail), and re-suspended in 0.6 ml RIPA buffer. Sonication was performed on a Misonic 3000 in a 15 ml centrifuge tube (Corning) submerged in 95% ethanol on dry ice with the following regimen: 6 x 30 sec at setting #7 with 1 min cooling intervals, 2 x 30 sec at setting #2.5 with 1 min cooling intervals, 4 x 30 sec at setting #5 with 1 min cooling intervals, and 30 sec at setting #7. Sonicates were transferred to 1.5 ml microcentrifuge tubes (Eppendorf) and spun at 14,000 RPM for 30 min at 4°C to pellet debris. Cleared supernatants were transferred to clean microcentrifuge tubes and their volumes were adjusted with RIPA buffer so that each 0.3 ml of sonicate contained chromatin from ~400,000 larvae. 1% of this chromatin was aliquoted, frozen and saved as an input control for sequencing. For each immunoprecipitation, 2 micrograms of the appropriate antibody was added to 0.3 ml of sonicate and incubated overnight at 4°C on a nutator. The following day, 150 microliters of magnetic beads with the appropriate immunoglobulin (Dynal/Invitrogen) were added and incubated for 3-4 hr on a nutator. Beads were collected with the Dynal magnet and the supernatants were saved. Beads were washed five times in LiCl Wash Buffer (100 mM Tris, 500 mM LiCl, 1% NP40, 1% Sodium Deoxycholate) for 3 min each, followed by a brief wash in TE. The beads were then suspended in TE, transferred to a clean microcentrifuge tube, and TE was removed. 300 microliters of IP Elution Buffer (1% SDS and 0.1 M NaHCO3) and 2 microliters of Proteinase K (20 mg/ml; Ambion) were added to the beads and they were incubated at 42°C for 1-2 hr with periodic re-suspension of the beads and then overnight at 65°C. Eluate was transferred to a clean tube, and a second elution was performed with 100 microliters IP Elution Buffer at 65°C for 1 hr, and the two were combined. Eluate DNA was extracted with phenol/chloroform/isoamyl alcohol followed by chloroform/isoamyl alcohol. 25 micrograms of GenElute Linear Polyacrylamide (Sigma) was added as carrier, and DNA was precipitated with ethanol. Precipitated DNA was dissolved in 100 microliters Tris (10 mM, pH 8.0) and interrogated by QPCR and Fluorometry (NanoDrop) using PicoGreen (Molecular Probes/Invitrogen).
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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 |
3055KAAXX_7
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| Data processing |
Data were analyzed using the Python package ERANGE. 6hr_Starved_S2 and 6hr_Starved_S2#2 were pooled prior to analysis and analyzed under the name 6hr_Starved_S2. The number of reads was counted over each gene model and over a 200 bp window spanning the most 5’ transcription start site for the input and each of the samples. Read density was computed from these counts by normalizing to reads per million per kilobase of sequence. The variance in input read density correlates with the variance in sample read density even after subtracting input read density from sample density, as if sequence-specific biases have a multiplicative effect during library preparation and sequencing. GC content is one possible explanation for this phenomenon. We therefore chose to divide sample read density for each region by input read density, rather than using subtraction, resulting in data with fold-enrichment (relative to input) as the unit. To avoid undefined and volatile ratios, we actually used the larger of the input read density or the median of all input read densities, making a conservative adjustment to fold-enrichment assessments. 5’ bias was defined as the ratio of fold-enrichment in the 200 bp window spanning the transcription start site to fold-enrichment over the gene model. To avoid undefined ratios in computing 5’ bias, fold-enrichments in the denominator (gene model) of zero were changed to 0.1.
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| Submission date |
Dec 15, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
L. Ryan Baugh |
| E-mail(s) |
ryan.baugh@duke.edu
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| Phone |
919-613-8179
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| Organization name |
Duke University
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| Department |
Biology
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| Lab |
Baugh
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| Street address |
4314 French Family Science Center
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708-0338 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (2) |
| GSE13973 |
ChIP-Seq of RNA Pol II with antibodies S2, 4H8 and 8WG16 during C. elegans L1 arrest and recovery |
| GSE14009 |
Nutritional control of gene expression during C. elegans L1 arrest and recovery |
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| Relations |
| SRA |
SRX003823 |
| BioSample |
SAMN02195627 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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