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Sample GSM1676267 Query DataSets for GSM1676267
Status Public on Jun 05, 2015
Title C. elegans RNA dauer rep 1
Sample type SRA
 
Source name whole organism
Organism Caenorhabditis elegans
Characteristics strain: N2 strain
developmental stage: dauer
Growth protocol S. carpocapsae (strain All), S. scapterisci (strain FL), S. feltiae (strain SN), S. glaseri (strain NC), and S. monticolum (Mount Jiri strain) were reared and maintained using standard methods (Kaya and Stock 1997). Briefly, 5 last-instar Galleria mellonella waxmoth larvae or a single adult cricket for S. scapterisci (American Cricket Ranch, Lakeside, CA) were placed in a 5 cm Petri dish with a 55 mm Whatman 1 filter paper acting as a pseudo-soil substrate in the bottom of the dish. ≤250 ml containing 500-1000 IJs suspended in water was evenly distributed on the filter paper. After 7-10 days the insect cadavers were placed on White traps (White 1927). Waxmoth cadavers infected with S. glaseri were placed in a petri dish partially filled with plaster of Paris and harvested from this, since S. glaseri emerge as pre-IJs that will not properly develop if they emerge directly into water (Kaya and Stock 1997). Emerging IJs from all species were harvested, washed for 10 minutes in 0.4% Hyamine 1622 solution (Fluka), and rinsed 3 times with water.
Extracted molecule total RNA
Extraction protocol To harvest bulk genomic DNA and IJ RNA, infective juveniles from each species were washed in a low percent Hyamine when collected, as above. They were then re-washed and collected in Ringer’s solution. After the last wash in Ringers, the worms were suspended in for 15- 30 minutes. For DNA extraction a Wizard® Genomic DNA Purification Kit (Promega) was used, following the manufacturers protocol. The genomic DNA was then treated with RNase A to remove any RNAs present in the sample. For RNA extractions, the nematodes were snap-frozen in liquid nitrogen in ~100-μL aliquots and stored at −80°C. Worms were then freeze-thawed three or four times to break the cuticle before extracting bulk RNA. Bulk RNA was then extracted using a Phenol-Chloroform extraction using Trizol® (Invitrogen). This sample was treated with DNAse to remove lingering DNA and then treated to poly-A selection to isolate Eukaryotic messenger RNA, reducing if not removing bacterial contaminant. To isolate embryonic, L1, and adult stage-specific RNA from S. carpocapsae, one to two thousand IJs were placed onto 10cm lipid agar plates seeded with overnight cultures of Xenorhabdus nematophila. These cultures were allowed to grow for ~42 hours to collect adults or 3 days to collect embryos and L1s. To collect embryos and L1s, gravid females were harvested from the 10cm plates 3 days after IJs were placed there. The females were harvested by adding enough distilled H2O to the cover the surface of the plates, which were then swirled by hand to lift the nematodes into suspension and pour them into conical tubes. These were then pelleted by gentle centrifugation and rinsed several times with distilled H2O until the supernatant was clear. The nematodes were then placed in separate conical tubes in 5ml aliquots. These were then topped of to 50ml with bleach solution (16.6ml of 12% bleach, 5ml of 1M KOH, and 80ml of distilled H2O). Eggs were harvested by bleaching the nematodes until all nematode tissue was dissolved, leaving only the eggs. These embryos were then either harvested for total RNA as described above or they were allowed to hatch in Ringers solution, where the L1s were then harvested for total RNA as described above. To isolate embryonic, L1, and adult stage-specific RNA from S. feltiae, one to two thousand IJs were placed onto 10 cm lipid agar plates seeded with overnight cultures of Xenorhabdus bovienii. These cultures were allowed to grow for ~36 hours to collect adults or ~55 hours to collect embryos and L1s. The same procedure was used to harvest embryos and L1s as S. carpocapsae. To isolate embryonic, L1, and adult stage-specific RNA from C. elegans N2s, worms were placed onto 10 cm NGM plates seeded with overnight cultures of Escherichia coli  (OP50 strain). 200 uL aliquots OP50 were added every day. Plates with lots of gravid adults were bleached according to the guide for Maintenance of C. elegans in Wormbook (Stiernagle, 2006). The embryos were either collected to harvest embryos, placed in Ringer’s solution for ~ 20 hours to harvest L1s, or plated on fresh NGM plates seeded with E. coli OP50 and collected ~47 hours  later to collect young adults.
The RNA-seq libraries used for genome annotation were built using an unstranded, paired-end library construction protocol (Mortazavi et al. 2008; Mortazavi et al. 2010) . Libraries were quantified using Qubit fluorometer (Invitrogen) and size distributions were verified using Agilent Bioanalyzer and the High Sensitivity DNA Kit. These RNA-seq libraries were sequenced on the Illumina Genome Analyzer IIx sequencer in paired-end mode to a read length of 76 nt. The RNA-seq libraries that were used for the gene expression analyses were built using a first-strand protocol, and sequenced on the NextSeq 500 sequencer in single-end mode to a read length of 75 nts.  
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description first-strand, single-end, read_length= 75 bp
Data processing Gene expression quantifiation: Stranded, single-ended RNA-seq library reads from the embryonic, L1, IJ, and adult stages of S. feltiae, S. carpocapsae, and C. elegans were trimmed to 35 bp to remove low quality bp. Prior to mapping the reads, indexes were prepared for each respective species annotations (C. elegans: ce10/WS220) using the RSEM command (version 1.2.12) rsem-prepare-reference. Reads were mapped to the respective species annotations using Bowtie 0.12.8 with the following options: -S --best, --strata, -k 10, -m 10, -v 1. Isoform expression was quantified using RSEM command, rsem-calculate-expression, with the following options: --bam, --fragment-length-mean. Isoform expression was summed to obtain the expression for each gene.
Genome_build: S. carpocapsae RNA-seq data was mapped to carpocapsae annotations from genome version 1: GCA_000757645.1 (http://www.ncbi.nlm.nih.gov/assembly/GCA_000757645.1/). Annotations can be found on Wormbase.
Genome_build: S. feltiae RNA-seq data was mapped to S. feltiae annotations from genome version 1: GCA_000757705.1 (http://www.ncbi.nlm.nih.gov/assembly/GCA_000757705.1). Annotations can be found on Wormbase.
Genome_build: C. elegans RNA-seq data was mapped to WS220 transcripts sequences.
Supplementary_files_format_and_content: Each sample expression file (*.isoforms.results) contains the isoform IDs, and the corresponding RPKMs, TPMs, and counts for each gene isoform.
Supplementary_files_format_and_content: S. carpocapsae gene identifier: L569_g1, S. carpocapsae isoform identifier: L569_g1.t1 (http://parasite.wormbase.org/Steinernema_carpocapsae_prjna202318/Info/Index)
Supplementary_files_format_and_content: S. feltiae gene identifier: L889_g1, S. feltiae isoform identifier: L889_g1.t1 (http://parasite.wormbase.org/Steinernema_feltiae_prjna204661/Info/Index)
 
Submission date May 05, 2015
Last update date May 15, 2019
Contact name Marissa Macchietto
E-mail(s) mmacchie@umn.edu
Organization name University of Minnesota, Minneapolis
Department Minnesota Supercomputing Institute
Street address 117 Pleasant Street SE
City Minneapolis
ZIP/Postal code 55455
Country USA
 
Platform ID GPL19757
Series (1)
GSE68588 Comparative genomics of Steinernema reveals deeply conserved gene regulatory networks
Relations
BioSample SAMN03604151
SRA SRX1019178

Supplementary file Size Download File type/resource
GSM1676267_EIJr1.isoforms.results.txt.gz 482.0 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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