title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of whole body L2 females, L2 hermaphrodites, L2 converted females (hermaphrodite-fated larvae induced to develop as females), adult males and mixed stages individuals from the three-sexed nematode Auanema rhodensis
design: RNA-seq of whole body L2 females, L2 hermaphrodites, L2 converted females (hermaphrodite-fated larvae induced to develop as females), adult males and mixed stages individuals from the three-sexed nematode Auanema rhodensis
library_construction_protocol: Converted-females L2 larvae were isolated by using synchronized progeny populations generated by hermaphrodite mothers. Briefly, dauers (fated to become hermaphrodites) were isolated and allowed to develop into adult hermaphrodites. After ~12 h of egg laying the mothers were removed and the early eggs laid were left to hatch. To convert hermaphrodite-fated larvae into females, mid-L1 larvae with smaller gonads (characteristic of hermaphrodite-fated L1s) were isolated and grown with E. coli streptoycin-resistant strain OP50-1 containing 200 nM dafachronic acid (DA) on Nematode Growth Medium (NGM) in individual wells of a 12 well culture plate. Once the larvae reached the L2 stage, the ones that had a female morphology were collected and used for RNA extraction. About 200 L2s were picked and transferred to an Eppendorf tube containing 200 µL of M9 buffer. The nematodes were washed 2-3 times in M9 buffer, allowing them to sink to the bottom of the tube under gravity between each wash. After the final wash, the maximum amount of M9 was removed and 200 µL of Trizol was added to the tube. The tube was placed at -80 °C immediately. For RNA extraction, nematodes were first freeze-cracked in liquid nitrogen (2-3 times). Trizol was added to make up the volume to 500 µL and nematodes were shaken with a few sterile 0.5 mm glass beads on a BeadBeater homogenizer (20 s, 3 times with 30 s intervals). Subsequently, RNA was extracted using a standard chloroform approach and the pellet was dissolved in DEPC treated water and stored at -80 °C. Illumina TruSeq RNA sample Prep kit and protocol was used to construct the mRNA-seq libraries. Briefly, the poly-A containing RNA molecules were purified using poly-T oligo-attached magnetic beads.  Following fragmentation of the mRNA, a first strand cDNA synthesis was performed using a reverse transcriptase and random primers, followed by a second strand cDNA synthesis. The cDNA fragments were then end repaired, an 'A' base was added, and finally the adapters were ligated. The resulting products were then purified twice and amplified by PCR to create the final cDNA library.
attributes.tag: Experimental Factor: sex
attributes.value: Converted-female