GSM6124908: HK104, 30ºC, replicate 2; Caenorhabditis briggsae; ncRNA-Seq
Total RNA from gravid adult hermaphrodites in mass isogenic cultures of each strain-temperature combination was isolated with TRIzol extraction and isopropanol precipitation. Using the Ambion MirVana kit, we separated the small RNA fractions <200 bp and then isolated small RNAs between 18 and 30 nucleotides using gel extraction from 15% acrylamide gels. The samples were treated with tobacco acid pyrophosphatase to convert triphosphorylated RNA into a monophosphorylated form prior to ligating the 3' linker, followed  by 15% polyacrylamide/7M urea gel extraction. A 5' linker sequence, possessing one of nine 4 nucleotide barcodes at its 3' end, was ligated to the samples to enable multiplexing. This was followed by reverse-transcription cDNA synthesis, and PCR amplification, to add Illumina sequencing adapters with an additional 8 nucleotide indexing barcode on the 3' adapter.