GSM7656673: biol rep 2 L3 larval stage (male) of dpy-28(y1);him-8(e1489) grown at 25oC; Caenorhabditis elegans; RNA-Seq
Worms pellet were collected in 500 µL of Trizol and flash frozen in liquid nitrogen and stored at -80oC until further processing. Total RNA was prepared from the frozen worm pellets following the instructions for the TRIzol LS (Invitrogen) protocol. After the isopropanol precipitation step, the RNA was re-suspended in the extraction buffer of the RNA isolation kit (PicoPure, Arcturus), and further isolation was carried out in accordance with the manufacturer's instructions. RNA libraries for RNA seq were prepared using a bulk version of the MARS-Seq procedure was employed. Briefly, reverse transcription was used to barcode and pool 18 ng of input RNA from each sample. The pooled samples underwent second strand synthesis after Agencourt Ampure XP beads cleanup (Beckman Coulter), and they were linearly amplified by T7 in vitro transcription. The resulting RNA was fragmented and then converted into final library by tagging the samples with Illumina sequences during ligation, RT, and PCR