GSM8375014: YL701, ETOH Control, 3 h, rep2; Caenorhabditis elegans; ncRNA-Seq
Samples were subjected to three freeze/thaw cycles (thawed for 2 min at 37 °C/flash frozen for 1 min). Samples were centrifuged for 5 min at 14800 g, and supernatants transferred to a new tube and mixed 1:1 with cold 100% ethanol. RNA isolation was performed with Direct-zol RNA microprep kit(Zymo Research #R2062). RNA was eluted with 15 μL of nuclease-free water (Zymo Research) and treated with 2 μLQuick CIP (NEB M0525S) in rCutSmart Buffer (NEB B6004S) ina total reaction volume of 20 μL at 37 °C for 90 min. CIP-treated RNA samples were heat-inactivated for 2 min at 80 °C, suspended in 100 μL Trizol (Invitrogen) and purified with Direct-zol RNA microprep kit (Zymo Research #R2062). The RNA was then treated with 1.5 μL RppH (NEB M0356S) in a total reaction volume of 20 μL for 1 h at 37 °C, resuspended in 100 μL Trizol(Invitrogen) and purified with Direct-zol RNA microprep kit (Zymo Research #R2062). The Yale Center for Genome Analysis (YCGA) prepared the small RNA library with the QiaSeq miRNA library prep kit (Qiagen) and performed sequencing on an Illumina NovaSeq using 75 bp paired-end sequencing. The RNA quality and fragment size distribution were confirmed with a Bioanalyzer. RNA libraries for small RNA-seq were prepared using Qiagen micro mRNA-Seq Library Prep Kit following manufacturer's protocols.