small RNA-seq of C. elegans, HT115 fed 25C
For each replicate, approximately 8,000 day 1 adult hermaphrodite worms were snap frozen and ground with a mortar and pestle for 15 minutes prior to trizol extraction of total RNA. Next, RNAs 15 to 30 nucleotides long were gel-purified from 20ug of total RNA on a 15% polyacrylamide/urea gel (Criterion). Small RNAs were then ligated to a 3 adaptor using truncated T4 RNA ligase 2 (New England BioLabs). To include 5-triphosphorylated RNAs in cloning, samples were next treated with Antarctic Phosphatase (New England BioLabs) and then treated with T4 polynucleotide kinase (New England BioLabs) as previously described (Gent et al, 2009). Adaptor-ligated small RNAs and any excess 3 adaptor were then hybridized to the oligo that would eventually be used as the reverse transcription primer prior to ligation of the 5 adaptor. This order of operations helps to minimize adaptor-dimer formation (McReynolds and Munafo, 2014). To help avoid cross-contamination, the 5 adaptor was modified to contain the Illumina genomic sequencing primer annealing site followed by an additional 4 nucleotides at the 3 end. Twelve different 5 adapters ending in unique 4 nucleotide long sequences were mixed in equal proportions, and this mixture was then ligated using T4 RNA ligase I (New England BioLabs). Every sample was treated with the same mix of 5 adapters. Libraries were each amplified and multiplexed with a unique 6-nucleotide long 3 barcode, then pooled for next-generation sequencing