RNASeq_DF5112_mixed_devstage
Strains were all cultured under the same conditions on Nematode Growth Medium (NGM) plates seeded with E. coli OP50 bacteria at 20C. Nematode cultures were grown until worms of all stages (embryos, larvae and gravid adults) were visible before bacterial food was exhausted and then processed for RNA extraction. Mixed worm populations were collected from plates by washing them off the plates with M9 medium, followed by 4 rounds of spinning and washing. Worm pellets were fast-frozen in 50-100 uL of water in liquid nitrogen and stored at -80oC. For RNA isolation 500 uL Trizol LS were added to the frozen pellet and the worms lysed with 6 freeze thaw cycles (~30 sec in liquid nitrogen, and 2 min in 37oC water bath, after each cycle samples were vortexed for 30 sec). Samples were spinned down at max speed for 2 min to pellet debris and corpses. The supernatant was transferred to a fresh tube. Then 100% ethanol in a 1:1 ratio was added, mixed well and pipetted into a Direct-zol RNA Miniprep kit (Zymo Research) column. Samples were processed according to the manufacturer's instructions including the in-column DNase digestion for 30 min. Total RNA was resuspended in 30 ul of RNase-free water. RNA integrity was tested by agarose gel electrophoresis and Bioanalyzer (RNA Nano Assay) and quantified using Qubit RNA HS Assay Kit in a Qubit 2.0 Fluorometer (Life technologies). NGS library prep was performed with Illumina's TruSeq stranded mRNA LT Sample Prep Kit following Illuminas standard protocol (Part # 15031047 Rev. E) using  of the reagents. Libraries were prepared with a starting amount of 250ng and amplified in 10 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies).