GSM9745551: 3376_25; Caenorhabditis elegans; RNA-Seq
Total RNA was isolated from day 1 adults (~72 hours following L1 synchronization) using standard phenol-chloroform method with TRIzolTM (Invitrogen). RNA quality was accessed using Agilent 4150 BioAnalyzer instrument and High Sensitivity RNA ScreenTapes. To sequence the mRNA, RNA libraries were prepared using NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® coupled with NEBNext® Poly(A) mRNA Magnetic Isolation Module. The cDNA libraries were pooled and paired-end sequencing was performed on the Nextseq 2000 platform.  For sRNA sequencing, we treated the RNA samples with RNA 5′ Polyphosphatase (epicentre) and the libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) or TruSeq Small RNA Library Prep Kit (Illumina) according to the manufacturer's protocol. RNA ranging from ~140 to 160 nt was size-selected by gel extraction using 4% agarose E-Gel (Life Technologies). The pooled samples were the sequenced using the Illumina HiSeq 2500 instrument.