GSM9471615: 1688_m01h93_1; Caenorhabditis elegans; RNA-Seq
For RNA extraction, synchronized gravid adult animals (~68 h at 20 °C after L1 arrest) were washed off plates with water three times and then settled on ice to clear bacteria. Worm pellet was resuspended in 1mL TRIzol reagent (Life Technologies) and freeze-thawed on dry ice followed by chloroform extraction and isopropanol precipitation. 40 ng of total RNA was used for total small RNA library preparation. Small RNAs (18 to 30-nt) were size selected on homemade 10% Urea-polyacrylamide gels. Small RNAs were treated with 5' RNA polyphosphatase (Epicenter RP8092H) and ligated to 3' pre-adenylated adapters with Truncated T4 RNA ligase (NEB M0373L). Small RNAs were then hybridized to the reverse transcription primer, ligated to the 5' adapter with T4 RNA ligase (NEB M0204L), and reverse transcribed with Superscript III (Thermo Fisher 18080–051). Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase (NEB M0491L) and size selected on a homemade 10% polyacrylamide gel. Library concentration was determined using the Qubit 1 X dsDNA HS Assay kit (Thermo Fisher Q33231) and quality was assessed using the Agilent BioAnalyzer. Libraries were sequenced on the Illumina NextSeq2000 (SE 75 bp reads) platform.