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
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
To obtain RNA from males, about 500 young adult males were picked in a 1.5 mL centrifuge tube containing 0.5 mL of M9 and washed twice with the buffer. M9 was then replaced with 0.5 mL Trizol and the tube frozen at -80 °C. 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.
Experimental Factor: sex
male