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| Status |
Public on Apr 25, 2012 |
| Title |
12hr_Starved BC5 |
| Sample type |
SRA |
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| Source name |
L1 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
develomental stage: L1 larvae
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| Treatment protocol |
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 at 20°C and 180 rpm for 65 hr and then bleached to produce a clean preparation of embryos. Embryos were suspended in S-complete at 5 eggs/mL and incubated at 20°C and 180 rpm for 24 hr so that they hatch and enter L1 arrest. After 24 hr, cultures were fed with 40 mg/mL HB101 to initiate synchronous development and they were incubated at 20°C and 180 rpm for 57 hr. After 57 hr the cultures were bleached, and the eggs were suspended in S-complete at 10 eggs/mL. In these conditions, the first eggs are fertilized at about 53 hr, and when bleached at 57 hr the yield is typically about 10 eggs per worm. Animals were incubated at 20°C and 180 rpm for 24 hr so that they hatch and enter L1 arrest. The 0 hr time point was collected 24 hr after bleaching, corresponding to approximately 12 hr L1 arrest. The remainder of the culture was fed with 25 mg/mL HB101 and incubated at 20°C and 180 rpm with collections at 1, 3 and 6 hr after feeding . Larvae were collected by centrifugation and washed 3 times in S-basal before being flash frozen. Lewis, J.A., and Fleming, John T. 1995. Basic Culture Methods. In Caenorhabditis elegans: Modern biological analysis of an organism (eds. H.F. Epstein, and Shakes, D. C.), pp. 4-27. Academic Press, San Diego, California.
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| Growth protocol |
Wild-type C. elegans strain N2 was used for RNA-seq. The N2 stock used was from the Sternberg collection at the California Institute of Technology; this strain was obtained from the Caenorhabditis Genetics Center in 1987, 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.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was prepared using TRIzol (Invitrogen) according to the manufacturer’s protocol with minor modifications. 1 mL TRIzol was used per sample, and homogenization was supplemented with 100 mL 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. 200 ng 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 approximately 100 ng RNA) was used for adapter ligation. cDNA was gel purified to capture inserts of RNA fragment size 100 – 200 nt. 12 PCR cycles were used to amplify the libraries. Libraries were processed and sequenced on the Solid 4 system according to the manufacturer’s protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
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| Description |
Reads mapping to genome and transcriptome and eads mapping to genome after 22 bp stripped from 5' end of read when the 22 bp is either SL1 or SL2
mRNA-seq: single-stranded RNA adapter ligation 5' sequencing
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| Data processing |
We first mapped reads to the WormBase 210 version of the genome (WS210) using Bowtie (v. 0.12.7) (Langmead et al. 2009). Reads that did not map in this step were aligned to the Wormbase 220 predicted transcriptome, whose coordinates had been mapped back to the WS210 coordinate system. We used Bowtie to map to the transcriptome as opposed to TopHat because we found that many reads mapped to transcriptome-derived splice junctions with Bowtie were not mapped with TopHat (data not shown). ~70% of genes in C. elegans are spliced to a 22 nt sequence donated by a snRNA called a ‘trans-splice leader’ (T Blumenthal 2005). The vast majority of trans-splice leaders come from two different sequences: either splice leader 1 or 2 (SL1 or SL2) (T Blumenthal 2005). We were interested in quantifying the overall levels of trans-splicing. Therefore, reads that did not map to either the genome or the transcriptome were stripped of the first (5’) 22 nt of sequence and remapped to determine if these reads came from the 5’ end of trans-spliced mRNAs. We determined whether the stripped 22 nt sequence corresponded to SL1 or SL2 for the reads that mapped after stripping. We created alignment files of reads mapping to the genome and transcriptome along with an index linking the IDs of reads containing splice leaders to the type of splice leader contained in the read (ie, SL1 or SL2). Langmead B, Trapnell C, Pop M, and Salzberg S. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10: R25. doi:10.1186/gb-2009-10-3-r25 Blumenthal, T. 2005. Trans-splicing and operons. In Wormbook (ed. The C. Elegans Research Community). http://www.wormbook.org.
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| Submission date |
Oct 17, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Colin Maxwell |
| E-mail(s) |
colin.maxwell@duke.edu
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| Organization name |
Duke
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| Department |
Biology
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| Lab |
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) |
| GSE33023 |
mRNA-seq of L1 larvae recovering from arrest |
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| Relations |
| SRA |
SRX101385 |
| BioSample |
SAMN00739402 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM818548_BC5.DEXSeq.count.txt.gz |
413.6 Kb |
(ftp)(http) |
TXT |
| GSM818548_BC5.GnTn.WS210.Ensembl.bam |
1.7 Gb |
(ftp)(http) |
BAM |
| GSM818548_BC5.genome.WS210.mapped.SL1_2.bam |
2.6 Mb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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