GSM3861115: IGN RNA-seq rep2; Caenorhabditis elegans; RNA-Seq
Growth:  Worms were grown to starvation on NGM plates. Starved L1s were floated on peptone-enriched plates, and grown gravid at 20Ë?C. Gravid adults were bleached and synchronized by L1 starvation.  L1s were plated to enriched plates and grown until the young adult stage. Extraction: Animals for nuclei isolation were collected from six enriched plates (~0.3 million) per RNA-seq experiment. Young adult animals were harvested at 54-56 hours after plating synchronized L1s when most of the animals had 4-10 embryos. Worms were collected into one 50 mL conical tube and spun at 3100 rpm for 2 minutes and then washed 3x in M9. Worms were incubated for 30â??45 minutes in M9 buffer with shaking after the third M9 wash to reduce intestinal bacteria. Then germ nuclei isolation was performed. 500 ÂµL TRIzol was added to the nuclei pellet.  Total RNA isolation was performed with standard TRIzol RNA extraction. Total RNA was then treated with DNA-free rDNase I and cleaned up using RNeasy Mini Kit. rRNA was depleted by Ribo-Zero rRNA Removal Kit. Library construction:  The Yale Center for Genome Analysis (YCGA) prepared libraries for each sample using the Kapa Biosystems reagents.