GSM6260366: spe44.r3_86A3; Caenorhabditis elegans; RNA-Seq
500 L4/young adult males were hand-picked into M9 + 0.05% gelatin, washed 1X and pelleted.  Liquid was removed down to 25ul and 500ul TRIzol (Invitrogen) was added.  The mixture incubated at room temperature for 5 minutes with periodic vortexing.  Carcasses were pelleted by centrifugation and supernatant was removed to a clean tube.  100 ul chloroform was added to the supernatant, vortexed for 30 seconds and phases separated by centrifugation at 15,000 RPM for 5 minutes.  250 ul of the upper aqueous phase was removed to a new tube and 350 ul chloroform was added, vortexed and separated as in the previous steps.  250 ul of isopropanol was added to the aqueous phase along with 1ul Glycoblue (Invitrogen) and vortexed.  The solution was centrifuged for 20 minutes at 15,000 RPM and the supernatant was decanted.  The pellet was washed with 1 ml 75% ethanol twice and dried at room temperature for 10 minutes.  The dried pellet was resuspended in 16 ul nuclease-free water. Concentration was determined with the NanoDrop. Using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 (BioO/PerkinElmer), polyA-selected RNA was isolated from 300 ng of total RNA according to manufacturer's instructions.  Sequencing libraries were prepared using the same kit according to manufacturer's instructions.