RNA-Seq of Caenorhabditis elegans; day 4, ergo, rep 1
For RNA extraction, worms were synchronized by bleaching, and maintained as previously described with few modifications. Worms were transferred every day using M9 buffer. Animals were collected from plates and washed at least three times with M9 using gravity to remove bacteria and progeny. After the last wash, worms were spread over the treatment or vehicle plates. On the day of collection, 80 animals were transferred into a 1.5 ml tube, washed three times with M9 and excess buffer was removed. 500 L of Trizol reagent was immediately added to the worm pellet and samples were flash frozen in liquid nitrogen. Samples were subjected to three freeze-thaw cycles (fast thawing in the water bath followed by 30 s vortexing and direct freezing in liquid nitrogen). After the third cycle, 200 uL of phenol/chloroform/ isoamyl alcohol mix (25/24/1, v/v/v) was added, samples were vigorously shaken by hand for 15 s, incubated for 2 min at RT and centrifuged at 12,000 x g for 15 min. The upper aqueous phase was carefully transferred to a new tube and mixed with an equal volume of 70% ethanol in RNAse free water. RNA purification was further performed using PureLink  RNA Mini Kit following manufacturers protocol. RNA quality and yield were checked using a NanoDrop spectrophotometer and immediately frozen at -80C. Sequencing was performed by the Beijing Genomic Institute (BGI, Hong Kong) using DNBSEQ sequencing technology.