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| Status |
Public on May 27, 2016 |
| Title |
Starved_L1_daf16_B |
| Sample type |
SRA |
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| Source name |
Arrested L1 larvae of strain GR1307 [daf-16(mgDf50)]
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: GR1307 [daf-16(mgDf50)]
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| Growth protocol |
C. elegans strain GR1307 [daf-16mgDf50)] was used for RNA-seq. This strain was obtained from the Caenorhabditis Genetics Center in March 2009, expanded, and frozen. Nematodes were maintained on standard NGM plates with E. coli OP50 as food (Lewis 1995), but liquid culture was used to prepare RNA-seq samples. A starved 5-cm plate was used to inoculate 25 mL of liquid culture (S-complete plus 40 mg/mL E. coli HB101) (Lewis 1995). The culture was incubated for 65 h at 20°C and 180 rpm and then bleached to produce a clean preparation of embryos. Embryos were suspended in S-complete at 5 eggs/μL and incubated for 24 h at 20°C and 180 rpm so that they hatch and enter L1 arrest. After 24 h, cultures were fed with 40 mg/mL HB101 to initiate synchronous development, and they were incubated for 57 h at 20°C and 180 rpm. In these conditions, the first eggs are fertilized at ∼53 h, and when bleached at 57 h the yield is typically about 10 eggs per worm. After 57 h, the cultures were bleached, and the eggs were suspended in S-complete at 10 eggs/μL. Animals were incubated for 24 h at 20°C and 180 rpm so that they hatch and enter L1 arrest. Larvae were collected by centrifugation and washed three times in S-basal before being flash-frozen. Two biological replicates were collected.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using TRIzol (Invitrogen) according to the manufacturer's protocol with minor modifications. One milliliter of TRIzol was used per sample, and homogenization was supplemented with 100 μL of acid-washed sand. Poly-adenylated mRNA was isolated from total RNA using Dynal oligo(dT) magnetic beads (Invitrogen) according to the manufacturer's protocol.
Two hundred nanograms of poly-adenylated RNA was used in a Tobacco Acid Pyrophosphatase reaction (Epicentre) according to the manufacturer's protocol in order to remove 5′ caps. The product was purified with phenol:chloroform extraction and ethanol precipitation using GlycoBlue as a coprecipitant (Ambion). This purified product was then used in the RNase III fragmentation reaction at the beginning of the Solid Total RNA-Seq Kit Whole Transcriptome protocol (Applied Biosystems). The manufacturer's instructions were followed for the remainder of the library preparation process. Fragmentation efficiency was analyzed with the BioAnalyzer (Agilent), and one-half of each sample (corresponding to ∼100 ng of RNA) was used for adapter ligation. cDNA was gel-purified to capture inserts of RNA fragment size 100–200 nt. Twelve PCR cycles were used to amplify the libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
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| Data processing |
Bowtie was used to map the reads to the WS210 genome in colorspace with the options "--best -k 1 -m 2 -S"
Reads were counted using hseq-count using WS220 annotations mapped to WS210 coordinates and the the options "-s yes"
Genome_build: WS210 with WS220 annotations
Supplementary_files_format_and_content: htseq-count files
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| Submission date |
May 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Colin Scott Maxwell |
| E-mail(s) |
[email protected]
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| Organization name |
Duke University
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| Department |
Biology
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| Lab |
L. Ryan Baugh
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| Street address |
4314 FFSC
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
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| Platform ID |
GPL14752 |
| Series (1) |
| GSE69329 |
dbl-1/TGF-β and daf-12/NHR signaling mediate cell-nonautonomous effects of daf-16/FOXO on starvation-induced developmental arrest |
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| Relations |
| BioSample |
SAMN03742200 |
| SRA |
SRX1041795 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1697878_smRNA3_BC12.combinedMapping.count.txt.gz |
134.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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