alias: GSM1676255
title: GSM1676255: S. feltiae RNA embryo rep 1; Steinernema feltiae; RNA-Seq
library_construction_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.  
identifiers.namespace: GEO
identifiers.id: GSM1676255